WO2011138409A1 - Combretastatin analogs for use in the treatment of cancer - Google Patents

Combretastatin analogs for use in the treatment of cancer Download PDF

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WO2011138409A1
WO2011138409A1 PCT/EP2011/057234 EP2011057234W WO2011138409A1 WO 2011138409 A1 WO2011138409 A1 WO 2011138409A1 EP 2011057234 W EP2011057234 W EP 2011057234W WO 2011138409 A1 WO2011138409 A1 WO 2011138409A1
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compound
cancer
mmol
methyl
compounds
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PCT/EP2011/057234
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French (fr)
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Rainer Schobert
Bernhard Biersack
Thomas Müller
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Universität Bayreuth
Martin-Luther-Universität Halle-Wittenberg
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Priority to JP2013508509A priority Critical patent/JP5636091B2/en
Priority to CN201180033235.5A priority patent/CN102985410B/en
Priority to US13/695,804 priority patent/US8980933B2/en
Priority to EP11723014.4A priority patent/EP2566851B1/en
Publication of WO2011138409A1 publication Critical patent/WO2011138409A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/41641,3-Diazoles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/41641,3-Diazoles
    • A61K31/41781,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42Oxazoles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61NELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00Radiation therapy
    • A61N5/10X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • A61P35/02Antineoplastic agents specific for leukemia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D233/00Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/56Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms
    • C07D233/58Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring nitrogen atoms
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D233/00Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
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    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D263/00Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/30Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D263/32Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D403/00Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07DHETEROCYCLIC COMPOUNDS
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    • C07D403/02Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/10Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings

Definitions

  • the present invention relates to specific analogs of combretastatin, in particular the compounds of formula (I) as defined herein, and pharmaceutical compositions comprising the compounds, as well as their medical use, in particular in the treatment or prevention of cancer, including multidrug-resistant cancer.
  • the chemotherapy of cancer is generally limited by the high incidence of malignant tumors that display resistance to a range of chemical anti-tumor agents, either intrinsically or acquired upon repeated administration.
  • chemotherapeutical agents capable of overcoming this so-called multidrug resistance.
  • An alternative therapy of such cases is not available, either.
  • a water-soluble phosphate prodrug of the angiotoxic, naturally occurring lead compound combretastatin A-4 (CA-4) has already entered clinical phase I! and IIS trials against various incurable cancer diseases.
  • CA-4 and analogs thereof are strongly cytotoxic and selectively disrupt tumoral vasculature or prevent its neoformation (so-called antivascular or antiangiogenic effect, respectively). They also bind to tubulin and inhibit its polymerization thus impeding the cell proliferation (antimitotic effect). In combination, these effects lead to an inhibition of tumor cell proliferation and of the growth and spread of solid tumors (invasion, metastasis).
  • CA-4 A drawback of CA-4 is its insufficient cytotoxicity which necessitates combination regimens with carboptatin or taxol in the therapy of solid tumors. Treatment with CA-4 alone often led to the persistence of peripheral cancer cells and hence to tumor relapses (Tron et al., J. Med. Chem. 2006, 49, 3033-3044; Lippert, Bioorg. Med. Chern. 2007, 15, 605-615).
  • the related CA-1 and its bisphosphate prodrug ⁇ 4503 were also spectacularly efficacious in certain tumor modeis owing to their catechoie moiety.
  • ROS reactive oxygen species
  • the compounds of formula (1), (I!) or (HI) as defined herein below have an improved efficacy against cancer, in particular against resistant tumor cells, and sufficient solubility in water or serum. They generally have a low toxicity which avoids cumulative dose restrictions and leads to improved tolerance. Furthermore, they have an improved chemical stability and thus reduced tendency to inactivation.
  • the present invention relates to a compound of formula (I)
  • X is selected from O, S, N(H), or N(Ci_4 alkyl).
  • X is selected from O or N ⁇ C H aikyl). More preferably, X is selected from O or N(CH 3 ).
  • R 1 is selected from halogen, -CN, -CF 3 , -NH 2 , -NH(C 1 _ 4 alkyl), or -N(C ⁇ alkyi)(C-,_4 alkyl).
  • R 1 is selected from halogen (such as, e.g., -F, -CI, or -Br), or -NH 2 . More preferably, R 1 is selected from -CI, -Br, or -NH 2 . Most preferably, R 1 is selected from -CI or -Br.
  • R 2 is selected from hydrogen, halogen, -CN, -CF 3 , -OH, -0(d_4 alkyl), -NH 2 , -NH(d_4 alkyl), or - (C W alkyl)(C M alkyl).
  • R 2 is selected from hydrogen, halogen (such as, e.g., -F, -CI, or -Br), -OH, or -NH 2 . More preferably, R 2 is selected from -F, -OH, or -NH 2 .
  • R 3 is selected from -OH, -0(C ⁇ alkyl), -SH, -S(C M alkyl), -NH 2l -NH(C ⁇ alkyl), or - N(Ci-4 alkyl)(Ci_4 alkyl).
  • R 3 is selected from -0(C 1-4 alkyl) or -N(C 1-4 alkylXd ⁇ alkyl). More preferably, R 3 is selected from -0-CH 3 , -0-CH 2 -CH 3 or ⁇ N(CH 3 ) 2 , particularly from -O-CH 3 or -N(CH 3 ) 2 .
  • the compound of formula (i) may have the foliowing structure:
  • halogen may, e.g., be -F, -CI, or -Br, and preferably is -CI, and the other groups X and R 1 are as defined above.
  • R 2 is -NH 2 and R 3 is -O-CH 3 .
  • X is O or N(CH 3 )
  • R 1 is -NH 2 or halogen (such as, e.g., -F, -CI, or -Br)
  • R 2 is -NH 2
  • R 3 is -O-CH 3 .
  • a preferred compound of formula (I) is 1-methyl-5-(3-amino-4- methoxyphenyl)-4-(3-chloro-4,5-dimethoxyphenyI)-imidazole (also referred to as "5b" or "compound 5b").
  • Another preferred compound of formula (I) is 1 -methyI-5-(3-amino-4- methoxyphenyl)-4-(3-bromo-4,5-dimethoxyphenyI)-imidazole (also referred to as “6b” or “compound 6b”).
  • R 2 is -NH 2 and R 3 is -0-CH 2 -CH 3 .
  • X is O or N(CH 3 )
  • R 1 is -NH 2 or halogen (such as, e.g., -F, -CI, or -Br)
  • R z is - NH 2
  • R 3 is -0-CH 2 -CH 3 .
  • Particularly preferred compounds of formula (!) are, accordingly, 1-methyl-5-(3-amino-4-ethoxyphenyi)-4-(3-chloro-4,5-dimethoxyphenyl)- imidazole (i.e., 1-methyl-5-(3"-amino-4"-ethoxyphenyl)-4-(3'-chloro-4',5 , -dimethoxyphenyl)- imidazole; also referred to as “5f or "compound 5f) and 1-methyl-5-(3-amino-4- ethoxyphenyl)-4-(3-bromo-4,5-dimethoxyphenyl)-imidazole (i.e., 1-methyl-5- ⁇ 3"-amino-4"- ethoxyphenyi)-4-(3'-bromo-4 ! ,5'-dimethoxyphenyi)-imidazole; also referred to as "6F or "compound 6f ).
  • R 2 is -F and R 3 is -0-CH 3 or -0-CH 2 -CH 3 .
  • X is O or N(CH 3 )
  • R 1 is -NH 2 or halogen (such as, e.g., -CI, or -Br)
  • R 2 is -F
  • R 3 is -0-CH 3 or -0-CH 2 -CH 3 .
  • preferred compounds of formula (I) are 1-methyl-4-(3-chloro-4,5-dimethoxyphenyl)-5-(3-fluoro-4-ethoxypheny!-imidazole (also referred to as “5g” or “compound 5g”) and 1-methyl-4-(3-bromo-4,5-dimethoxyphenyl) ⁇ 5-(3-fiuoro-4-ethoxyphenyl)-imidazole (also referred to as "6g” or “compound “6g”).
  • the present invention further provides a compound of formula (II)
  • X 2 Is selected from O, S, N(H), or N(C 1-4 alkyl).
  • X 2 is selected from O or N ⁇ Ci_4 alkyl). More preferably, X 2 is selected from O or N(CH 3 ).
  • R 21 is selected from halogen, -CN, -CF 3 , -NH 2 , -NH(C 1-4 alkyl), or -N(C 1-4 a!kyiXd ⁇ alkyl).
  • R 21 is selected from halogen (such as, e.g., -F, -CI, or -Br), or -NH 2 . More preferably, R 21 is selected from -CI, -Br, or -NH 2 . Most preferably, R 21 is selected from -CI or -Br.
  • R 22 is Ci_4 aikyi.
  • R 22 is C 2 _ aikyl. More preferably, R 22 is ethyi.
  • halogen comprised in the compound of formula (II) is preferably selected from - F, -CI, or -Br, and more preferably the "halogen" is -CI.
  • Preferred compounds of formula (II) are 1 -methyl-4-(3-amino-4,5-dimethoxyphenyl)-5-(N- methyl-3-chloroindol-5-yl)-imidazole, l -methyl ⁇ -iS'-chloro ⁇ '.S'-dimethoxyphenylJ-S-iN- methyl-3"-chloroindol-5 ,, -yi)-imidazole, and l-meihyi ⁇ -iS'-bromo ⁇ S'-dimethoxyphenyiy-S- (N-methyl-3"-chloroindoi-5"-y!)-imidazole.
  • a particularly preferred compound of formula (II) is 1-methyl-4-(3'-bromo-4',5 , -dimethoxyphenyl)-5-(N-ethyi-3"-chloroindo!-5"-yl)-imidazole (also referred to as “6h” or “compound 6h”).
  • the invention also provides a compound of formula (ill)
  • X 3 is selected from O, S, N(H), or (C M alkyl).
  • X 3 is selected from O or N(C 1-4 alkyl). More preferabiy, X 3 is selected from O or N(CH 3 ).
  • R 3 is selected from halogen (particularly -CI, -Br or -I), -CN, -CF 3 , -NH 2 , - HfC ⁇ alky!), or - (d_ alkyi)(Ci_4 alkyl).
  • R 3 is selected from -Ci, -Br, -I, or -NH 2 . More preferably, R 31 is selected from -Br, -I, or -NH 2 . Most preferably, R 31 is selected from -Br or -I.
  • R 32 is selected from hydrogen, halogen, -CN, -CF 3 , -OH, -0(C ⁇ alkyl), -NH 2 , -NH(C- alky!), or -N(C H a!kyi)(C 1 _ 4 alkyl).
  • R 32 is selected from hydrogen, halogen (such as, e.g., -F, -CI, or -Br), -OH, or -NH 2 . More preferably, R 32 is selected from -F, -OH, or -NH 2 . Most preferably, R 32 is -F.
  • R 33 is selected from -OH, -0(C 1-4 alkyl), -SH, -S ⁇ d ⁇ alkyl), -NH 2 , -NH(C M alkyl), or - N(C!_4 alkyt)(C _4 alkyl).
  • R 33 is selected from -0(0 1-4 alkyl) or -N(C 1 _ 4 alkyI)(C 1 _ 4 alkyl). More preferably, R 33 is selected from ⁇ 0-CH 3s TM0-CH 2 -CH 3 or -N(CH 3 ) 2 . Most preferably, R 33 is selected from -0-CH 3 or -0-CH 2 -CH 3 .
  • the C 1-4 alkyl comprised in that group is preferably selected from methyl or ethyl.
  • FT 1 is selected from -OH, -0(C W alkyl), -SH, -S(d_4 alkyl), halogen (particularly -CI, -Br or -I), -CN, -CF 3 , -NH 2 , -NH(C ⁇ a!kyl), or -N(C ⁇ aikyl)(C M alkyl).
  • R 34 is selected from -0(C ⁇ alkyl), -CI, -Br, -I, or -NH 2 . More preferably, R 34 is selected from -O- CHs, -0-CH 2 -CH 3l -Br, -I, or -NH 2 .
  • R 34 is selected from -0-CH 3 , -Br or - I.
  • R is -F and R is -0-CH 3 .
  • X 3 is N(CH 3 )
  • R 31 is -Br or -I
  • R 32 is -F
  • R 33 is -0-CH 3
  • R 34 is -0-CH 3 , -Br or -I.
  • Particularly preferred compounds of formula (lii) are the following compounds 9a, 9b and 9c:
  • alkyl refers to a monovalent saturated aliphatic (i.e. non-aromatic) acyclic hydrocarbon group (i.e. a group consisting of carbon atoms and hydrogen atoms) which may be linear or branched and does not comprise any carbon-to-carbon double bond or any carbon-to-carbon triple bond. Accordingly, the term “C ⁇ alky! refers to methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, tert-butyl, or sec-butyl).
  • halogen refers to -F, -CI, -Br, or -I, and in particular to -F, -CI, or -Br.
  • the present invention also relates to a pharmaceutical composition comprising a compound of formula (I), (II) or (III) as defined herein or a pharmaceutically acceptable salt, solvate, or prodrug thereof, in combination with a pharmaceuticaliy acceptable excipient. Accordingly, the compounds of formula (I), (II) or (III) are useful as medicaments.
  • the present invention further relates to a compound of formula (I), (II) or (III) as defined herein, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, or a pharmaceutical composition comprising any of the aforementioned entities in combination with a pharmaceutically acceptable excipient, for use in the treatment or prevention of cancer.
  • a pharmaceutically acceptable salt, solvate, or prodrug thereof or a pharmaceutical composition comprising any of the aforementioned entities in combination with a pharmaceutically acceptable excipient, for use in the treatment or prevention of cancer.
  • the invention encompasses a method of treating or preventing cancer, the method comprising the administration of a compound of formula (I), (II) or (III) as defined herein, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, or a pharmaceutical composition comprising any of the aforementioned entities in combination with a pharmaceutically acceptable excipient, to a subject in need of such a treatment or prevention.
  • the cancer to be treated or prevented with the compounds or the pharmaceutical compositions according to the present invention includes, for example, breast (mamma) cancer, genitourinary cancer (such as, e.g., prostate tumor, including a hormone-refractory prostate tumor, or germ cell cancer), lung cancer (such as, e.g., small cell or non-small cell lung tumor), gastrointestinal cancer (such as, e.g., hepatocellular carcinoma, colorectal tumor, colon cancer or gastric cancer), epidermoid cancer (such as, e.g., epidermoid head and/or neck tumor or mouth tumor), melanoma, ovarian cancer, pancreas cancer, neuroblastoma, head and/or neck cancer, bladder cancer, renal cancer, brain cancer, leukemia (such as, e.g., lymphocytic leukemia or myelogenous leukemia), or lymphoma.
  • breast (mamma) cancer such as, e.g., prostate tumor, including
  • the cancer to be treated or prevented with the compounds or the pharmaceutical compositions according to the invention is a multidrug-resistant cancer.
  • the cancer to be treated may thus be a multidrug-resistant form of the above described cancers.
  • the cancer to be treated or prevented with the compounds or the pharmaceutical compositions provided herein is resistant against com breta statin A-4 and/or cisplatin. Accordingly, in the present invention the treatment or prevention of combretastatin A-4 (CA-4)-refractory cancer or cisplatin- refractory cancer is particularly envisaged.
  • the compounds according to the invention have been found to be surprisingly effective in the medical intervention of multidrug-resistant cancer and, in particular, of CA4- refractory cancer, which has been demonstrated in vitro and in vivo, including in a mouse xenograft model (cf. Example 3 and Figures 3, 4 and 5). It has been demonstrated that the new compounds exhibit a distinct cytotoxic, antiangiogenic/antivascular and antimitotic effect.
  • any isomerization to a trans-stilben with loss of anti-tumor activity is no longer possible.
  • their polar substituents render the new compounds very well soluble in water and so directly applicable without employment of a vehicle.
  • These compounds can be administered by injection as solutions in physiological saline solution. Such solutions were shown to be stable for at least one month under ambient conditions without any noticeable loss of activity.
  • animal studies of a multi-resistant tumor model the new compounds were found anti-tumor active causing a dramatic regression of the tumor xenografts.
  • the strong antivascular effect selectively affecting the tumoral vasculature was unambiguously confirmed by visible hemorrhages of the tumoral tissue. No unwanted side effects were observed when the new compounds were applied at efficacious concentrations.
  • the bromo imidazole 6b was vascular disrupting in tumor xenografts while leaving regular vasculature in chicken embryos (CAM assay) alone.
  • the compounds of the invention are also characterized by an outstanding tumor-selective cytotoxicity and a strong induction of cancer cell apoptosis.
  • the compounds of the invention further have advantageous properties with respect to solubility in water and chemical stability in solution, as also demonstrated in Example 3 and Figures 1 and 2.
  • solubility of the compounds 5b, 6b and 8a according to the present invention in water exceeded a concentration of 10 mg/mL
  • these compounds are even more water-soluble than the potassium salt of combretastatin A-4-phosphate (about 5 mg/mL; Bedford et a!., Bioorg. Med. Chem. Lett. 1996, 6, 157-160).
  • the compounds of the invention, including the compounds of formula (I) are particularly suitable and effective as medicaments, including as medicaments in the treatment or prevention of cancer.
  • the compounds of the present invention are particularly advantageous in respect of cytotoxicity, solubility in water, and chemical stability, as has been shown in the appended examples for the compound 5b according to the invention.
  • compound 5b is highly cytotoxic in vitro and in vivo (likewise in CA-4- and cisplatin-resistant cell Sines), shows an improved water solubility compared with CA-4-phosphate and can not isomerize to inactive frans-isomers iike CA-4.
  • Compound 5b is nevertheless well tolerated and selective in mouse xenografts.
  • the compounds of the present invention can thus be administered without the need of prodrug forms, such as phosphate prodrug forms used for combretastatin-A4.
  • prodrugs can be used, if desired.
  • the compounds of formula (I) can be prepared according to the following general protocol. First, the respective para-toluenesu!fonylmethylisocyanides (Tos iC derivatives) are prepared (Scheme 1 ).
  • Scheme 1 Synthesis of para-toluenesuifonylmethySisocyanides 4. Reagents and conditions: (i) CH 3 I, K 2 C0 3 , TBAi, DMF, 20°C, 24h, 80-90%; (ii) HCONH 2 , camphorsulfonic acid, para-toluenesulfinic acid, 60°C, 16h, 51-58%; (iii) POCl 3 , Et 3 N, D E, -5°C, 3h, 57-74%.
  • halo-substituted Tos iC derivatives 4a/b are converted to the imidazoles 5/6 by reaction in dimethoxyethane/ethano! mixtures with aryl aldehydes or the imines generated from the latter (Scheme 2), Selective reduction of the nitro group with Zn/HCI affords the desired amines 5b or 6b, respectively.
  • Treatment of the imidazoles 5a- and 6b-d with 3 M HCl/dioxane yields the respective water-solub!e hydrochlorides.
  • R2 H
  • R 3 N(CH 3 ) 2
  • the nitro-substituted compounds 7a-e are prepared similarly to the imidazoles 5/6 from the nitro-TosMIC derivative 4c and the corresponding aldehydes and imines (Scheme 3).
  • the amines 8a-e are obtained by Pd-catalyzed transfer hydrogenation.
  • the 3-chloroindole 7e is reduced with Zn/HCI to amine 8e.
  • the compounds 8 can further be converted to water-solubie hydrochlorides.
  • the compounds 5e-g and 6e-g are prepared as follows (Scheme 4). 4-Ethoxy- 3-nitro/fluorobenza!dehyde is treated with MeNH 2 to give imine intermediates, which are reacted with the TosMIC reagents 4a/b under basic conditions to give the N-methyl imidazoles 5e,g and 6e,g.
  • the preparation of the amines 5f and 6f is accomplished by reduction of 5e/6e with Zn/HCI in THF. These compounds can be converted into the hydrochloride salts, e.g., by treatment with 3 M HCI/dioxane.
  • the compounds of formula (I) can also be prepared in analogy to the synthesis routes described in Wang et a!., J. Med. Chem. 2002, 45, 1697-171 1.
  • the compounds of formula (II) or (111) can be prepared in accordance with or in anaiogy to the syntheses described above and/or in the Examples,
  • the compounds of formula (ill), in particular, can also be prepared according to the fo!iowing general protocol.
  • the compounds 9a-c are prepared as fo!lows (Scheme 5).
  • the scope of the invention embraces all pharmaceutically acceptable salt forms of the compounds of the present invention, in particular the compounds of formula (I), (II) or (III), which may be formed, e.g., by protonatton of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of a carboxylic acid group with a physio!ogically acceptable cation as they are well known in the art.
  • Exemplary base addition salts comprise, for example, alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; ammonium salts; aliphatic amine salts such as trimethyiamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamtne, procaine salts, meglumine salts, diethanol amine salts or ethylenediamine salts; aralkyl amine salts such as ⁇ , ⁇ -dibenzy!ethylenediamine salts, benetamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts or isoquinoiine salts; quaternary ammonium salts such as tetramethyiammonium salts, tetraethylammonium salts, benzyltrimethylarnmonium salts, benzyltriethylammoni
  • Exemplary acid addition salts comprise, for example, mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts, nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dthydrogenphosphate salts), carbonate salts, hydrogencarbonate salts or perchlorate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, undecanoate, lactate, maleate, oxalate, fumarate, tartrate, maiate, citrate, nicotinate, benzoate, salicylate or ascorbate salts; sulfonate salts such as methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulf
  • the scope of the invention embraces solid forms of the compounds of formula (I), (II) or (111) in any soivated form, including e.g. solvates with water, for example hydrates, or with organic solvents such as, e.g., methanol, ethanol or acetonitriie, i.e. as a methanolate, ethanolate or acetonitrilate, respectively; or in the form of any polymorph.
  • all stereoisomers of the compounds of formula (I), (II) or (111) are contemplated as part of the present invention, either in admixture or in pure or substantially pure form.
  • the scope of the compounds according to the invention embraces all the possible stereoisomers and their mixtures. It particularly embraces the racemic forms and the isolated optical isomers.
  • the racemic forms can be resolved by physical methods, such as, e.g., fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chirai column chromatography.
  • the individual optical isomers can be obtained from the racemates using conventional methods, such as, e.g., salt formation with an optically active acid followed by crystallization.
  • prodrugs of the compounds of formula (!), (II) or (III) are derivatives which have chemically or metabolicai!y cleavable groups and become, by soivolysis or under physiological conditions, the compounds of the present invention which are pharmaceutically active in vivo.
  • Prodrugs of the compounds of formula (I), (II) or (III) may be formed in a conventional manner with a functional group of the compounds such as with an amino or a hydroxy! group.
  • the prodrug derivative form often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgaard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985).
  • an acyloxy derivative prepared by reacting the hydroxyl group with a suitable acylhalide or a suitable acid anhydride is exemplified as a prodrug.
  • an amide derivative prepared by reacting the amino group with a suitable acid halide or a suitable mixed anhydride is exemplified as a prodrug.
  • the compounds described herein may be administered as compounds per se or may be formulated as medicaments.
  • the medicaments/pharmaceutical compositions may optionally comprise one or more pharmaceutically acceptable excipients, such as carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, or solubility enhancers.
  • the pharmaceutical compositions may comprise one or more solubility enhancers, such as, e.g., poly(ethy!ene glycol), including poly ⁇ ethylene glycol) having a molecular weight in the range of about 200 to about 5,000 Da, ethylene glycol, propylene glycol, non-ionic surfactants, tyloxapol, polysorbate 80, macrogol-15-hydroxystearate, phospholipids, lecithin, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, cyc!odextrins, hydroxyethyl-p-cyclodextrin, hydroxypropyl- ⁇ - cyclodextrin, hydroxyethyi-Y-cycSodextrin, hydroxypropyl-y-cyclodextrin, dihydroxypropyl- ⁇ - cyclodextrin, glucosyl
  • compositions can be formulated by techniques known to the person skilled in the art, such as the techniques published in Remington's Pharmaceutical Sciences, 20 th Edition.
  • the pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as intramuscular, intravenous, intraperitoneal, subcutaneous, intradermal, intraarterial, rectal, nasal, topical, aerosol or vaginal administration.
  • Dosage forms for oral administration include coated and uncoated tablets, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets.
  • Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions and powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration.
  • Dosage forms for rectal and vaginal administration include suppositories and ovula.
  • Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler.
  • Dosage forms for topical administration include creams, gels, ointments, salves, patches and transdermal delivery systems.
  • the compounds of formula (I), (II) or (111) or the above described pharmaceutical compositions comprising a compound of formula (I), (I! or (111) may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to one or more of: oral (e.g. as a tablet, capsule, or as an ingestible solution), topical (e.g., transdermal, intranasal, ocular, buccal, and sublingual), parenteral (e. g., using injection techniques or infusion techniques, and including, for example, by injection, e.g.
  • oral e.g. as a tablet, capsule, or as an ingestible solution
  • topical e.g., transdermal, intranasal, ocular, buccal, and sublingual
  • parenteral e. g., using injection techniques or infusion techniques, and including, for example, by injection, e.g.
  • implant of a depot for example, subcutaneously or intramuscularly
  • pulmonary e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g. through mouth or nose
  • the compounds or the pharmaceutical compositions may be administered oraliy or by inhalation. It is particularly preferred that the compounds of the present invention are administered by pulmonary administration, in particular by inhalation, in the form of dry powder formulations as described e.g. in more detail herein below.
  • the compounds or pharmaceutical compositions are administered parenterally, then examples of such administration include one or more of: intravenously, intraarteriai!y, intraperitoneal, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the compounds pharmaceutical compositions, and/or by using infusion techniques.
  • parenteral administration the compounds are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • compositions under sterile conditions are readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
  • the compounds or pharmaceutical compositions can also be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
  • the tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disint eg rants such as starch (preferably com, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylceilulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
  • disint eg rants such as starch (preferably com, potato or tapioca starch), sodium starch glycolate, cro
  • Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • Preferred excipients in this regard include lactose, starch, a cellulose, miik sugar or high molecular weight polyethylene glycols.
  • the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanot, propylene glycol and glycerin, and combinations thereof.
  • the compounds or pharmaceutical compositions can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder.
  • the compounds of the present invention may also be derma!ly or transdermal administered, for example, by the use of a skin patch.
  • the compounds or pharmaceutical compositions may also be administered by the pulmonary route, rectal routes, or the ocular route.
  • they can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.
  • a preservative such as a benzylalkonium chloride.
  • they may be formulated in an ointment such as petrolatum.
  • dry powder formulations of the compounds of the present invention for pulmonary administration.
  • the dry powder formulations of the compounds of the present invention may be delivered using any suitable dry powder inhaler (DPI), i.e., an inhaler device that utilizes the patient's inhaled breath as a vehicle to transport the dry powder drug to the lungs.
  • DPI dry powder inhaler
  • the compounds or pharmaceutical compositions can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propyiene glycol, emulsifying wax and water.
  • they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, 2-octyldodecanol, benzyl alcohol and water.
  • a physician will determine the actual dosage which will be most suitable for an individual subject.
  • the specific dose level and frequency of dosage for any particular individual subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual subject undergoing therapy.
  • a proposed, yet non-limiting dose of the compounds of the invention, including the compounds of formula (I), (II) or (III), for administration to a human (of approximateiy 70 kg body weight) may be 0.1 pg to 10 g, preferably 0.1 mg to 1 g, and more preferably about 200 mg or more (e.g., 200 to 300 mg or 200 to 500 mg), of the active ingredient per unit dose.
  • the unit dose may be administered, for example, 1 to 4 times per week.
  • the dose will depend on the route of administration. It will be appreciated that it may be necessary to make routine variations to the dosage depending on the age and weight of the patient/subject as well as the severity of the condition to be treated. The precise dose and route of administration will ultimately be at the discretion of the attendant physician or veterinarian.
  • the compounds according to the present invention can be used in combination with other therapeutic agents.
  • the dose of each compound may differ from that when the compound is used alone.
  • the combination of a compound of this invention with (an) other drug(s) may comprise the administration of the drug(s) with the compound of the invention.
  • Such an administration may comprise simultaneous/concomitant administration. However, sequentiai/separate administration is also envisaged.
  • the second therapeutic agent to be administered in combination with the compound of the present invention is an anticancer drug.
  • the anticancer drug to be administered in combination with the compound of the invention may be: a tumor angiogenesis inhibitor (for example, a protease inhibitor, an epidermal growth factor receptor kinase inhibitor, or a vascular endothelial growth factor receptor kinase inhibitor); a cytotoxic drug (for example, an antimetabolite, such as purine and pyrimidine analogue antimetabolites); an antimitotic agent (for example, a microtubule stabilizing drug or an antimitotic alkaloid); a platinum coordination complex; an anti-tumor antibiotic; an alkylating agent (for example, a nitrogen mustard or a nitrosourea); an endocrine agent (for example, an adrenocorticosteroid, an androgen, an anti-androgen, an estrogen, an anti-esirogen, an aromatase inhibitor, a gonadotrop
  • An alkylating agent which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a nitrogen mustard (such as cyclophosphamide, mechiorethamine (chlormethine), uramustine, melphalan, chiorambucil, ifosfamide, bendamustine, or trofosfamide), a nitrosourea (such as carmustine, streptozocin, fotemustine, lomustine, nimustine, prednimusiine, ranimustine, or semustine), an aikyi sulfonate (such as busulfan, mannosulfan, or treosulfan), an aziridine (such as hexamethyimelamine (altretamine), triethyienemelamine, ThioTEPA ( ⁇ , ⁇ ' ⁇ '- triethylenethiophosphoramide), carboquone, or triaziquone), a hydrazin
  • a piatinum coordination compiex which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, or triplatin tetranitrate.
  • a cytotoxic drug which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, an antimetabolite, inciuding folic acid analogue antimetabolites (such as aminopterin, methotrexate, pemetrexed, or raltitrexed), purine analogue antimetabolites (such as ctadnbine, clofarabine, fludarabine, 6-mercaptopurine (including its prodrug form azathioprine), pentostatin, or 6-thioguanine), and pyrimidine analogue antimetabolites (such as cytarabine, decitabine, 5-fluorouraciS (including its prodrug forms capecitabine and tegafur), floxuridine, gemcitabine, enocitabine, or sapacitabine).
  • folic acid analogue antimetabolites such as aminopterin, methotrexate, pemetrexed, or raltitrexed
  • An antimitotic agent which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a taxane (such as docetaxei, iarotaxel, ortataxel, paclitaxel/taxoi, or tesetaxel), a Vinca alkaloid (such as vinblastine, vincristine, vinflunine, vindesine, or vinore!bine), an epothilone (such as epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, or epothilone F) or an epothilone B analogue (such as ixabepilone/azaepothilone B).
  • a taxane such as docetaxei, iarotaxel, ortataxel, paclitaxel/taxoi, or tesetaxel
  • a Vinca alkaloid such as vin
  • An anti-tumor antibiotic which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, an anthracycline (such as aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, or zorubicin), an anthracenedione (such as mitoxantrone, or pixantrone) or an anti-tumor antibiotic isolated from Streptomyces (such as actinomycin (including actinomycin D), bleomycin, mitomycin (including mitomycin C), or plicamycin).
  • an anthracycline such as aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, or zorubicin
  • a tyrosine kinase inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, axitinib, bosutinib, cediranib, dasatinib, eriotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxanib, sorafenib, sunitinib, or vandetanib.
  • a topoisomerase-inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a topoisomerase I inhibitor (such as irinotecan, topotecan, camptothecin, be!otecan, rubitecan, or lamellarin D) or a topoisomerase II inhibitor (such as amsacrine, etoposide, etoposide phosphate, teniposide, or doxorubicin).
  • a topoisomerase I inhibitor such as irinotecan, topotecan, camptothecin, be!otecan, rubitecan, or lamellarin D
  • a topoisomerase II inhibitor such as amsacrine, etoposide, etoposide phosphate, teniposide, or doxorubicin.
  • anticancer drugs may be used in combination with a compound of the present invention.
  • the anticancer drugs may comprise biological or chemical moiecules, like TNF- related apoptosis-inducing ligand (TRAIL), tamoxifen, amsacrine, bexarotene, estramustine, irofulven, trabectedin, cetuximab, panitumumab, tositumomab, alemtuzumab, bevacizumab, edrecolomab, gemtuzurnab, alvocidib, seiiciclib, aminolevulinic acid, methyl aminolevulinate, efaproxiral, porfimer sodium, talaporfin, temoporfin, verteporfin, alitretinoin, tretinoin, anagrelide, arsenic trioxide, atrasentan, bortezomib,
  • biological drugs like antibodies, antibody fragments, antibody constructs (for example, single-chain constructs), and/or modified antibodies (like CDR-grafted antibodies, humanized antibodies, "full humanized” antibodies, etc.) directed against cancer or tumor markers/factors/cytokines involved in proliferative diseases can be employed in co-therapy approaches with the compounds of the invention.
  • biological molecules are anti-HER2 antibodies (e.g. trastuzumab, Herceptin®), anti-CD20 antibodies (e.g.
  • Rituximab Rituxan ® , abThera ® , Reditux ®
  • anti-CD19/CD3 constructs see, e.g., EP-A-1 071 752
  • anti-TNF antibodies see, e.g., Taylor et ai., Antibody therapy for rheumatoid arthritis, Curr Opin Pharmacol, 2003, 3(3), 323-328.
  • the combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formuiation.
  • the individual components of such combinations may be administered either sequentially or simultaneously/concomitantly in separate or combined pharmaceutical formulations by any convenient route.
  • administration is sequential, either the present compound or the second therapeutic agent may be administered first.
  • administration is simultaneous, the combination may be administered either in the same or different pharmaceutical composition.
  • the two compounds When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation.
  • they may be provided in any convenient formulation, conveniently in such manner as are known for such compounds in the art.
  • the compounds of the present invention are administered in combination with physical therapy, such as radiotherapy.
  • Radiotherapy may commence before, after, or simultaneously with administration of the compounds.
  • radiotherapy may commence 1 to 10 minutes, 1 to 10 hours or 24 to 72 hours after administration of the compounds.
  • these time frames are not to be construed as limiting.
  • the subject is exposed to radiation, preferably gamma radiation, whereby the radiation may be provided in a single dose or in multiple doses that are administered over several hours, days and/or weeks.
  • Gamma radiation may be delivered according to standard radiotherapeutic protocols using standard dosages and regimens.
  • the compounds of the present invention may be used to render cells, in particular undesired protiferative/hyperpro!iferative ceils like cancer or tumor cells, more susceptible to such a physical therapy, e.g. radiotherapy.
  • the present invention relates to a compounds of formula (I), (il) or (III) or a pharmaceutically acceptable salt, solvate, or prodrug thereof, or a pharmaceutical composition comprising any of the aforementioned entities in combination with a pharmaceutically acceptable excipient, for use in the treatment or prevention of cancer, whereby the compound or the pharmaceutical composition is to be administered in combination with an anti-proliferative drug, an anticancer drug, a cytostatic drug, a cytotoxic drug and/or radiotherapy.
  • the subject or patient may be an animal, a vertebrate animal, a mammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), a murine (e.g. a mouse), a canine (e.g. a dog), a feline (e.g. a cat), an equine (e.g. a horse), a primate, a simian (e.g. a monkey or ape), a monkey (e.g. a marmoset, a baboon), an ape (e. g.
  • a rodent e.g. a guinea pig, a hamster, a rat, a mouse
  • a murine e.g. a mouse
  • a canine e.g. a dog
  • a feline e.g. a cat
  • an equine e.g. a
  • mice gorilla, chimpanzee, orangutang, gibbon
  • a human The meaning of the terms "animal”, “mammal”, etc. is well known in the art and can, for example, be deduced from Wehner und Gehring (1995; Thieme Verlag).
  • animals are to be treated which are economically, agronomically or scientifically important.
  • Scientifically important organisms include, but are not limited to, mice, rats, rabbits, fruit flies like Drosophila melagonaster and nematodes like Caenorhabditis elegans.
  • Non-limiting examples of agronomically important animals are sheep, cattle and pig, whiie, for example, cats and dogs may be considered as economically important animals.
  • the subject/patient is a mammal. More preferably, the subject/patient is a human.
  • Treatment of a disorder or disease as used herein, e.g., in the case of the treatment of cancer, is well known in the art.
  • Treatment of a disorder or disease implies that a disorder or disease has been diagnosed in a patient/subject.
  • a patient/subject suspected of suffering from a disorder or disease typically shows specific clinical and/or pathological symptoms which a skilled person can easily attribute to a specific pathological condition (i.e. make a diagnosis of a disorder or disease).
  • Treatment of a disorder or disease may, for example, !ead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only).
  • Treatment of a disorder or disease may also lead to a partial response (e.g., ameiioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease.
  • “Amelioration" of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse, it is to be understood that a subject/patient may experience a broad range of responses to a treatment (e.g., the exemplary responses as described herein above).
  • Treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment ⁇ including symptomatic relief).
  • prevention of a disorder or disease is well known in the art.
  • a patient/subject suspected of being prone to suffer from a disorder or disease as defined herein may, in particular, benefit from a prevention of the disorder or disease.
  • the subject/patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition.
  • Such a predisposition can be determined by standard assays, using, for example, genetic markers or phenotypic indicators.
  • a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in the patient/subject (for example, the patient/subject does not show any clinical or pathological symptoms).
  • prevention comprises the use of compounds of the present invention before any clinical and/or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.
  • Figure 1 Inhibition of tubulin polymerization by 5b (freshly prepared solution vs solution having been stored for one month). Compound 5b effectively inhibits the polymerization of tubulin. No loss of activity is observed over time.
  • Test Tubulin-Polymerization assay.
  • Figure 2 Cytotoxic activity of 5b (freshly prepared solution vs solution having been stored for one month). The persistence of efficacy is proof of the chemical stability of 5b when dissolved (Test: SRB-cytotoxicity assay).
  • Figure 3 Comparison of the in vitro cytotoxicities of 5b and cispiatin (A) or of 5b, 6b and cisplatin (B). Treatment with 5b or 6b results in a breach of the multi-drug resistance of 141 1 HP cells (Test: SRB-cytotoxicity assay).
  • Figure 4 In vivo anti-tumor activity of compounds 5b and 6b in mouse xenografts of multi- resistant germ cell tumor eel! Sine 141 1 HP. The tumor response following sing!e-dose (A) and dual-dose (B) applications of test compounds, and the tumor response to repeated applications (C) are shown. Arrows indicate the administration of test compounds.
  • FIG. 5 Vascular disrupting effect of 5b.
  • the distinct vascular disrupting effect selectively affecting the tumoral vasculature is apparent from hemorrhages leading to red-blue to brown coloring of the entire tumor (A: before treatment; B: 24 hours after start of treatment) (Test: subcutaneous tumor xenografts in a nude mouse model; mice were anesthesized for the imaging).
  • Figure 6 Cell growth inhibition by reference compound 25f and the compounds 5b, 6b, 8a and 8e or 5c, 5d, 6c, 6d, 8b, 8c and 8d according to the invention (structures are indicated in Example 2 or 4) at various concentrations ⁇ : 100 ⁇ ; A : 1 ⁇ ; V : 0.01 ⁇ ; 0.001 ⁇ ) in ceils of human 518A2 melanoma, HL-60 leukemia, HT-29 colon adenocarcinoma, KB- V1/Vbl cervix carcinoma and MCF-7/Topo breast adenocarcinoma (A) or in cells of human 5 8A2 melanoma, HL-60 leukemia and HT-29 colon adenocarcinoma (B) upon incubation for 24 - 72 h (x-axis). Y-axis shows number of viable cells relative to untreated controls (1 ) as ascertained by the MTT assay.
  • Figure 7 Ceil growth inhibition by combretastatin A-4 at various concentrations ( ⁇ : 100 ⁇ ; A : 1 ⁇ ; #: 0.01 ⁇ ; ⁇ : 0.001 ⁇ ) in cells of human HT-29 colon adenocarcinoma, incubation for 24-72 h (x-axis). Y-axis shows number of viable cells relative to untreated controls as ascertained by the MTT assay.
  • Figure 8 Microscopic images of HL-60 cells tested in TUNEL assays after 16 h incubation with 10 ⁇ of the compound 5b, 6b or 8a.
  • the brightfie!d pictures show all the cells in the focus, the pictures of the green fluorescent channel (right) just the apoptotic cells, represented as bright dots.
  • FIG. 9 Chicken embryos with surrounding blood vessels immediately after adding the test compounds CA-4 or 5b (left), after one day (middle) and after three days (right) are shown. The top row shows a negative controi. Pictures are representative of at least two independent runs.
  • Figure 10 In vivo anti-tumor activity of compound 6b in mouse xenograft of multi-resistant germ cell tumor cell line 1411 HP. The tumor response following dual-dose application is shown. Arrows indicate the administration of test compound.
  • FIG 11 Vascular disrupting effect of compounds 5b, 6b, 5f and 6f in A2780 ovarian carcinoma xenograft tumors (A) and 1411 HP germ cell tumor xenografts (B).
  • the distinct vascular disrupting effect selectively affecting the tumoral vasculature is apparent from hemorrhages leading to red-blue to brown coloring of the entire tumor (day 0: before treatment; day 1 : 24 hours after start of treatment) (Test: subcutaneous tumor xenografts in a nude mouse model; mice were anesthesized for the imaging).
  • Figure 12 Vascular disrupting effect in 1411 HP germ cell tumor xenograft after oral administration of 6b.
  • the distinct vascular disrupting effect selectively affecting the tumoral vasculature is apparent from hemorrhages leading to red-blue to brown coloring of the entire tumor (day 0: before treatment; day 1 : 24 hours after start of treaiment) (Test: subcutaneous tumor xenografts in a nude mouse model; mice were anesthesized for the imaging).
  • Figure 13 Comparison of the in vitro cytotoxicities of compounds 5b, 6b, 5f and 6f compared to 25f in H12.1 germ cell tumor (A), 141 1 HP germ cell tumor (B), A2780 ovarian carcinoma (C), HT29 colon carcinoma (D), DLD1 colon carcinoma (E) and HCT8 colon carcinoma (F) cell lines (Test: SRB-cytotoxtcity assay).
  • Figure 14 Tube formation assay with HUVEC cells after treaiment with compound 8a (A), 6b (B) or 5b (C) at concentrations of 7.72 ng/mL or control (methanol) (D).
  • FIG. 15 High-content analysis of compound 6b (denoted as “Brimamin (Schobert)”) and compound 8a (denoted as “Amoxamin_2 (Schobert)”) in PtK-2 cells.
  • Example 1 Synthesis of 1 -methyl-5-(3-amino-4-methoxyphenyi)-4-(3-chioro-4,5- dimethoxyphenyl)-imidazole bis(hydrochloride) (5b x 2 HCI)
  • compound 5c was prepared from 3-fluoro-4- methoxybenzaldehyde (65 mg, 0.42 mmol), 33% MeNH 2 /ethanol (260 uL, 2.10 mmol) and acetic acid (150 ⁇ ) in boiling ethanol (15 mL) giving the imine intermediate, which was treated with compound 4a (153 mg, 0.42 mmol), dissolved in DME (10 mL), and K 2 C0 3 (500 mg, 3.62 mmol). After workup, the residue was purified by column chromatography (silica gel 60).
  • compound 5i Analogously to the synthesis of 5a, compound 5i was obtained from the imine of W-rnethyl-3- chloroindol-5-carboxaldehyde (81 mg, 0.42 mmoi), 33% MeNH 2 /ethanol (260 ⁇ _, 2.10 mmol) and acetic acid (150 ⁇ _) in boiling ethanoi (15 ml_), which was treated with 4a (153 mg, 0.42 mmol) and K 2 C0 3 (500 mg, 3.62 mmoi).
  • compound 6c was prepared from 3-fluoro-4- methoxybenzaldehyde (65 mg, 0.42 mmol), 33% MeNH 2 /ethanoi (260 pL, 2.10 mmol) and acetic acid (150 pL) in boiling ethanol (15 mL) giving the imine intermediate, which was treated with compound 4b (172 mg, 0.42 mmol), dissolved in DME (10 mL), and K 2 C0 3 (500 mg, 3.62 mmol). After workup, the residue was purified by column chromatography (silica gel 60; elution with ethyl acetate to 5% methanol/ethyl acetate).
  • compound 6d was prepared from 4-N,N- dimethyiaminobenzaldehyde (63 mg, 0.42 mmol), 33% eNH 2 /ethanol (260 pL, 2.10 mmoi) and acetic acid (150 ⁇ _) in boiling ethanoi (15 mL) giving the tmine intermediate, which was treated with compound 4b (172 mg, 0.42 mmol), dissolved in DME (10 mL), and K 2 C0 3 (500 mg, 3.62 mmol). After workup, the residue was purified by column chromatography (silica gel 60).
  • compound 6i was obtained from the imine of W-methyl-3- chloroindol-5-carboxaldehyde (81 mg, 0.42 mmo!), 33% MeNH 2 /ethanol (260 pL, 2.10 mmol) and acetic acid (150 pL) in boiling ethanol (15 mL), which was treated with 4b (170 mg, 0.42 mmol) and K 2 C0 3 (500 mg, 3.62 mmol). After workup the residue was purified by column chromatography (silica gel 60).
  • N-Methyl-3-chloroindol-5-carbaldehyde 7e' A/-Methylindol-5-carbaldehyde (400 mg, 2.5 mmol) was dissolved in dry acetonitrile (10 mL) and treated with /V-chiorosuccinimide (400 mg, 3.02 mmoi), whereupon the solution turned red.
  • the reaction mixture was stirred at room temperature for 20 h.
  • the solvent was removed in vacuum and the residue was purified by column chromatography (silica gel 60). Yield: 300 mg (1.55 mmol, 62%); colorless soiSd of mp 109 °C; f?
  • Example 3 Biological assays 1-Methyi-5-(3-amino-4-methoxyphenyl)-4-(3-chioro-4,5-dtmethoxyphenyl)-imidazole (“5b") as a representative compound of the present invention has been subjected to in vitro and in vivo assays, as described in the following.
  • the assay is fluorescence- based and tubulin polymerization was followed by measuring RFU (relative fluorescence units) on the SpectraFluorPlus (Tecan, Switzerland) using the following filters: excitation 360 nm, emission 465 nm.
  • the cytotoxic activity of 5b was established by standard colorimetric tests (SRB- and MTT- assays) against various tumor cell lines of different entities.
  • SRB cytotoxicity assay dose-response curves of the testicular germ cell tumor cell lines exposed to drug concentrations of 0.001-10 ⁇ were established using the sulforhodamine-B (SRB) microculture colorimetric assay (Papazisis et al. Optimization of the sulforhodamine B colorimetric assay. J, Immunol. Methods 1997, 208, 151 -158) and performed as described in: Muller et al.
  • mice were kept under pathogen-free conditions, fed on an autoclaved standard diet and given free access to sterilized water. Each of five mice were administered a 150 ⁇ 1_ phosphate buffered saline suspension of 10 million 141 1 HP cells into the left flank to generate subcutaneous xenograft tumors. After 4 weeks one group of two mice bearing 141 1 HP xenografts with a volume of ca. 1 cm 3 were injected i.p. with a single dose of 30 mg/kg body weight of 5b or 6b, respectively. Two mice in the second group with xenografts of a volume of ca. 2.5 cm 3 were injected i.p. with 20 mg/kg body weight of 5b on two consecutive days. Tumor volumes were calculated by caliper measurement using the formula a 2 * b * 0.5 with a being the short and b the Song dimension. Body weight was assessed twice weekly and daily while under therapy.
  • the compounds of formula (I), such as, e.g., compound 5b or 6b, represent a class of effectives suitable for the treatment of multi- resistant tumors. Their hallmark is the synergistic combination of high cancer ce!l-specific cytotoxicity based upon a genuine mechanism, a tumor-selective vascular disrupting effect, favourable pharmacological properties, and excellent tolerance in vivo.
  • the compounds 5f, 6b and 6f were further tested for vascular disrupting effect in the 141 1 HP xenograft model, and the compounds 5b, 5f, 6b and 6f were additionally tested in a second tumor model of A2780 ovarian carcinoma xenografts, showtng vascular disrupting activity of all compounds in both models ( Figure 1 1 ), which suggests a general activity of the compounds of the invention in well vascularized tumors.
  • Compounds 5b, 6b and 6f were also tested in terms of feasibility of oral administration. Mice received a single dose of 40 mg/kg body weight of 5b, 6b or 6f, respectively. No signs of toxicity were observed.
  • the vascular disrupting effect was studied 24h after giving 60mg/kg body weight of compound 6b peroral. No signs of toxicity were observed. The vascular disrupting effect occurred to same extent as seen after i.p. application, as also shown in Figure 12.
  • Example 4 Cell growth inhibition assay in various human tumor cell lines
  • the HL-60 cells were obtained from the German Collection of Biological Material (DS Z), Braunschweig, Germany; the human 518A2 melanoma cells as well as the testicular germ cell tumor eel! lines H12.1 and 1411 HP were cultured in the department of onco!ogy and hematology, Medical Faculty of the Martin-Luther University, Halie, Germany; the KBV1/Vbl and the MCF-7/Topo cells were obtained from the Institute of Pharmacy of the University Regensburg, Germany; and the colon HT-29 cells from the University Hospital Eriere, Germany.
  • the HL-60 and the HT-29 cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 lU/mL penicillin G, 100 pg/mL streptomycin sulfate, 0.25 pg/mL amphotericin B and 250 pg/mL geniamycine (all from Gibco, Egenstein, Germany).
  • FCS fetal calf serum
  • penicillin G 100 lU/mL
  • streptomycin sulfate 100 pg/mL streptomycin sulfate
  • 0.25 pg/mL amphotericin B 0.25 pg/mL amphotericin B
  • geniamycine all from Gibco, Egenstein, Germany.
  • the 518A2 and the KB-V1/Vbl cells were cultured in Dulbecco's Modified Eagle Medium (D-MEM, Gibco) containing 10% FCS, 100 lU/mL penicillin G, 100 pg/mL streptomycin sulfate, 0,25 pg/mL amphotericin B and 250 pg/mL gentamycine.
  • D-MEM Dulbecco's Modified Eagle Medium
  • FCS fetal U/mL
  • the MCF-7/Topo cells were grown in E-MEM medium (Sigma) supplemented with 2.2 g/L NaHC03, 1 0 mg/L sodium pyruvate and 5% FCS.
  • the cells were maintained in a moisture- saturated atmosphere (5% C0 2 ) at 37 °C in 75-mL culture flasks (Nunc, Wiesbaden, Germany). They were serially passaged following trypsinisation by 0.05% trypsin / 0.02% EDTA (PAA laboratories, Colbe, Germany). Mycoplasma contamination was routinely monitored, and only mycoplasma-free cultures were used.
  • MTT [3-(4,5-dimethy!thiazol-2-yl)-2,5-dipheny!tetrazolium bromide] (ABCR) was used to identify viable ceils which reduce it to a violet formazan (Mosmann, J. Immunol, Methods 1983, 65, 55-63).
  • HL-60 leukemia cells (5x 0 5 /mL), and cells (5x10 4 /mL) of 518A2 melanoma, HT-29 colon, KB-VWbl cervix and MCF-7/Topo breast carcinoma were seeded out in 96-well tissue culture plates and cultured for 24 h.
  • micropiates were swiftiy turned to discard the medium before adding the solvent mixture.
  • the micropiates were gently shaken in the dark for 30 min and absorbance at 570 nm and 630 nm (background) was measured with an ELISA plate reader.
  • AN experiments were carried out in quadruplicate; the percentage of viable cells was calculated as the mean ⁇ SD with controls set to 100%.
  • the halo-amino substituted imidazoles 5b and 6b were distinctly more cytotoxic than the known reference compound 25f at iC 5 o concentrations in the single- digit nanomolar range, even in the combretastatin A-4 resistant HT-29 cells and the Pgp- overexpressing KB-V1 cells. Only in the MCF-7/Topo cells they were noticeably less active but still superior to the known imidazole 25f.
  • diamino substituted oxazole 8a and its imidazole counterpart 8e were more cell line specific with greater efficacy than 25f and 5b/6b against both multidrug-resistant cells, i.e., KB-V1/Vbl (Pgp+) and MCF-7/Topo (BCRP ⁇ ). This is a hint at potentially different or additional modes of action of these compounds.
  • combretastatin A-4 shows lower cytotoxicities than compounds 5b and 6b in the multi-drug resistant HT-29 colon cancer cells. Hence, compounds 5b and 6b are able to overcome the drug resistance in these cancer cells in vitro.
  • the compounds 5b, 6b, 5f and 6f as well as the reference compound 25f were furthermore subjected to an SRB -cytotoxicity assay in a panel of tumor cell lines (H12.1 germ cell tumor, 141 1 HP germ ceil tumor, A2780 ovarian carcinoma, HT29 colon carcinoma, DLD1 colon carcinoma, HCT8 colon carcinoma).
  • SRB -cytotoxicity assay in a panel of tumor cell lines (H12.1 germ cell tumor, 141 1 HP germ ceil tumor, A2780 ovarian carcinoma, HT29 colon carcinoma, DLD1 colon carcinoma, HCT8 colon carcinoma).
  • compounds 5b and 6b showed superior activity as compared to compound 25f, and a further improvement of cytotoxicity was observed for the compounds 5f and 6f.
  • the compounds 9a, 9b and 9c which are exemplary compounds of formula (ill) according to the present invention, as well as the reference compound 25f were tested in a similar manner. As indicated in Table 1 , compounds 9a, 9b and 9c showed a more pronounced and selective cytotoxic effect on tumor cells (L929 fibroblasts, KB-3-1 cervix carcinoma, and PC-3 prostata cancer) than on non-malign cells (PtK-2 opossum kidney cells and NHDF fibroblasts) as well as an improved effect (MIC) on HUVEC cells as compared to the reference compound 25f.
  • tumor cells L929 fibroblasts, KB-3-1 cervix carcinoma, and PC-3 prostata cancer
  • non-malign cells PtK-2 opossum kidney cells and NHDF fibroblasts
  • MIC improved effect
  • Table 1 IC50 (nM) in L929 fibroblasts, KB-3-1 cervix carcinoma, PC-3 prostata cancer,
  • ROS reactive oxygen species
  • HL-60 cells 0.5'10 6 /mL were plated in 96-weli tissue culture plates, and test compounds were added after 24 h incubation at 37 °C to achieve a finai concentration of 50 ⁇ . Incubation (5% C0 2 , 95% humidity, 37°C) of cells following treatment with the test compounds was continued for 24 h. After removal of the cell medium by centrifugation, the cells in each well were resuspended in 100 pL 0.1 % NBT, and the plates were placed in the incubator for 1 h. The reduced NBT was solubilized with 100 ⁇ 2 M KOH and 130 pi DMSO for 30 min.
  • the absorbance was measured for each well at 630 and 405 nm (background) using an ELISA plate reader.
  • the adherent 518A2 cells (0.5- 0 4 /mL) were seeded out in 96- well tissue culture plates after trypsinization and incubation for 24 h at 37 °C to allow attachment, then treated similarly, only that the medium was removed prior to incubation with NBT for 4 h. All experiments were carried out in quadruplicate. The following results were obtained, the indicated values representing means of four independent experiments ⁇ standard deviation;
  • the extent of apoptosis-reiated mitochondrial damage in 518A2 and HL-60 cells was ascertained by means of the fluorescence dye JC-1 that detects changes in the mitochondrial membrane potential (Desager et aS. J. Cell. Biol. 1999, 144, 891-901 ).
  • the ratio of red to green fluorescence relative to untreated controls (100%) after 72 h exposure of 518A2 and HL-60 ceils to 5 ⁇ of the test compounds (i.e., compounds according to the invention or reference compound 25f) was determined with the Mitochondrial Membrane Detection Kit (Stratagene, La Jotla, CA, USA). Values represent means of four independent experiments ⁇ standard deviation.
  • TUNEL assays which allow the detection of apoptosis by iabel!ing the 3'-OH ends of DNA fragments with fiuorescein-tagged nucleotides, the compounds 5b, 6b and 8a were found to induce death in HL-60 cells predominantly in an apoptotic way (about 60% after 16 h incubation with 10 ⁇ drug).
  • the extent of apoptosis-related mitochondrial damage in HL-60 cells was ascertained by means of the fluorescence dye JC-1 that detects changes in the mitochondrial membrane potential (Desager et a!., J. Cell. Biol. 1999, 144, 891-901 ).
  • HL-60 ceils were incubated with the test compounds for 16 h, aliquots of 3 x 10 6 cells were withdrawn and washed/centrifuged 3 times in 200 pL PBS. The cells were fixed for 10 min at room temperature by suspending in 200 pL of a freshly prepared solution of 2% formalin in PBS, After washing with 2 x 200 pL PBS, 10 pL of the cell suspension was applied onto a microscope slide and air-dried at room temperature. The ceils were washed by covering with PBS for 5 min and treated for 2 min with a solution of 0.1 % Triton X-100 in 0.1% sodium citrate on ice.
  • TUNEL reaction mixture consisting of 1 pL TUNEL-Enzyme solution and 9 pL TUNEL-Label solution, was dropped on the cells which were then covered and incubated (5% C0 2 , 95% humidity) in the dark at 37 °C for 45 min.
  • the cells were washed three times with PBS and then analysed by fluorescence microscopy at an excitation wavelength of 450-500 nm. The percentage of apoptotic, TUNEL-posttive, green-stained cells was counted and calculated for 300 cells and expressed as mean ⁇ S.D. of three independent experiments.
  • Example 8 CAM assay Compounds of the present invention were tested for anti-angiogenic and vasculature disrupting properties using the CAM assay. In this test the vascular system of a fertilized chicken embryo is used as a model (Wilting et a!., Anat. Embryol. 1991 , 183, 259-271 ).
  • Fertilized chicken eggs received from a nearby farm directly after laying have been incubated at a temperature of 36-38 °C and a relative humidity of 60%. During the growth the eggs have been held in an inclined position and turned from time to time in order to avoid an adherence to the shell. After four days each embryo was transferred into a cavity created by fixing a thin plastic foil on top of a cup and covered. This was done by opening the shell at the flat end where the air sac resides and letting the content slip out. There the growth continued for another 2-4 days until the first blood vessels became visible. Then 10 nmol of the substance to be tested (in 10 pL PBS with 1 % DMF) have been applied directly on the embryonal vessels. As a reference PBS was used. Finally incubation continued for up to another three days (Dugan et al., Anat Rec. 1991 , 229, 125-128; Fisher, Tested studies for laboratory teaching 1993, 5, 105-1 15).
  • 35 ⁇ !_ of media matrigel (1 :1 ) were added into wells of a 96-well plate and incubated at 37 °C for 30 minutes. A trypsinized HUVEC cell suspension was set to 800.000 ceils/mL.
  • 25 ⁇ _ of the compound stock were serially diluted with 25 ⁇ _ of media.
  • 25 ⁇ _ of compound dilutions and 25 ⁇ !_ of cell suspension were added to the matrigel coated 96-well plate and the cells were incubated over night.
  • methanol was used as in place of the compound dilutions.
  • MWCS-moduie using DAP! (W1 ), FITC (W2) and TRSTC (W3) staining and filters.
  • Descriptors of MWCS-module e.g., MWCS-moduie using DAP! (W1 ), FITC (W2) and TRSTC (W3) staining and filters.
  • Image-based analysis total celis, (%) positive W2 W3, scoring profile 1-/12-/1-3/123. absolute number of celis appearing stained only at wavelengths 1 and 2 but not 3 (12-)
  • Cell-based analysis total area (area of nuclei), stained area W1/W2/W3 (stained area for individual dyes), positive W2/W3 (absolute numbers of stained celis at each wavelength); average/integrated intensity W1/W2 W3

Abstract

The present invention relates to specific analogs of combretastatin, in particular the compounds of formula (I) as described and defined herein, and pharmaceutical compositions comprising the compounds, as well as their medical use, in particular in the treatment or prevention of cancer, including multidrug-resistant cancer.

Description

COMBRETASTATIN ANALOGS FOR USE IN THE TREATMENT OF CANCER
The present invention relates to specific analogs of combretastatin, in particular the compounds of formula (I) as defined herein, and pharmaceutical compositions comprising the compounds, as well as their medical use, in particular in the treatment or prevention of cancer, including multidrug-resistant cancer.
The chemotherapy of cancer is generally limited by the high incidence of malignant tumors that display resistance to a range of chemical anti-tumor agents, either intrinsically or acquired upon repeated administration. Currently, there are no clinically established chemotherapeutical agents capable of overcoming this so-called multidrug resistance. An alternative therapy of such cases is not available, either. A water-soluble phosphate prodrug of the angiotoxic, naturally occurring lead compound combretastatin A-4 (CA-4) has already entered clinical phase I! and IIS trials against various incurable cancer diseases.
Figure imgf000002_0001
CA-4 and analogs thereof are strongly cytotoxic and selectively disrupt tumoral vasculature or prevent its neoformation (so-called antivascular or antiangiogenic effect, respectively). They also bind to tubulin and inhibit its polymerization thus impeding the cell proliferation (antimitotic effect). In combination, these effects lead to an inhibition of tumor cell proliferation and of the growth and spread of solid tumors (invasion, metastasis).
A drawback of CA-4 is its insufficient cytotoxicity which necessitates combination regimens with carboptatin or taxol in the therapy of solid tumors. Treatment with CA-4 alone often led to the persistence of peripheral cancer cells and hence to tumor relapses (Tron et al., J. Med. Chem. 2006, 49, 3033-3044; Lippert, Bioorg. Med. Chern. 2007, 15, 605-615). The related CA-1 and its bisphosphate prodrug ΟΧΪ4503 were also impressively efficacious in certain tumor modeis owing to their catechoie moiety. Catecholes are known to undergo redox cycling via quinoid intermediates thus mediating the generation of reactive oxygen species (ROS) and the alkylation of bionucleophiles (Ho!well et al., Anticancer Res. 2002, 22, 3933- 40; Folkes et al., Chem. Res. Toxicol. 2007, 20, 1885-1894).
The problem of insufficient cytotoxicity of CA-4 in vivo was overcome by developing combretastatin A-1 diphosphate which has a direct impact on cancer cells. However, like CA-4 this compound is prone to isomerization and thus deactivation. A preclinical study revealed that this isomerization can be precluded by replacing the olefin bridge by five- membered heterocycfes such as oxazoies or imidazoles (Wang et al., J. Med. Chem. 2002, 45, 1697-171 1 ). The resulting products are characterized by an improved water solubility and applicability in vivo without the need of prodrug formulations. In another study CA-4 analogous 3-halostilbenes were shown to have an enhanced affinity for tubulin and a more selective profile of efficacy (Pettit et al., J. Nat. Prod. 2005, 68, 1450-1458). Similar compounds are furthermore disclosed in: WO 01/09103; Vasiievsky et al, Chemistry of Heterocyclic Compounds, 2008, 44(10), 1257-1261 ; Orsini et al., Natural stilbenes and analoga as antineoplastic agents, in: Atta-ur-Rahman, Studies in Natural Products Chemistry, Bioactive Natural Products, Part N, 2008, Volume 34; Brown et al., Top. Heterocycl. Chem. 2006, 2, 1-51 ; Kiss LE et al., J Med Chem, 2010, 53(8), 3396-41 1 ; and Ohsumi K et al., Bioorg Med Chem Lett, 1998, 8{22), 3153-8.
Compounds resembling those prepared by Wang et al. or Pettit et al. fulfil only some rather than all of the requirements for optimum anti-tumor efficacy. Derivatives reminiscent of those described by Wang et ai. feature favourable pharmacological properties yet diminished tubulin affinity and cytotoxicities {ca. 1/100 th of that of CA-4) that are insufficient to treat resistant tumors effectively. On the other hand, compounds with increased cytotoxicity showed unfavourable pharmacological properties and lacked antivascular efficacy and activity against resistant tumors. Among the 3-halocombretastatin-A derivatives disclosed by Pettit et ai. there are some with distinct cytotoxic properties. However, owing to their stilbene nature and their lack of a stabilizing heterocyclic bridge, they tend to isomerize with loss of anticancer activity. In addition, their solubility in water and thus their applicability in vivo is rather limited unless converted into a phosphate ester. They were not amenable to tests with in vivo models. Moreover, the efficacy of the compounds described in Wang et al. and in Pettit et al. in the treatment of multi-resistant cancer and, in particular, in the treatment of CA-4- refractory cancer has not been examined. Hence, there is a strong demand for improved methods for the treatment or prevention of cancer, in particular mu!tidrug-resistant cancer. The compounds of formula (1), (I!) or (HI) as defined herein below have an improved efficacy against cancer, in particular against resistant tumor cells, and sufficient solubility in water or serum. They generally have a low toxicity which avoids cumulative dose restrictions and leads to improved tolerance. Furthermore, they have an improved chemical stability and thus reduced tendency to inactivation.
Accordingly, the present invention relates to a compound of formula (I)
Figure imgf000004_0001
(Q or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
X is selected from O, S, N(H), or N(Ci_4 alkyl). Preferably, X is selected from O or N{CH aikyl). More preferably, X is selected from O or N(CH3).
R1 is selected from halogen, -CN, -CF3, -NH2, -NH(C1_4 alkyl), or -N(C^ alkyi)(C-,_4 alkyl). Preferably, R1 is selected from halogen (such as, e.g., -F, -CI, or -Br), or -NH2. More preferably, R1 is selected from -CI, -Br, or -NH2. Most preferably, R1 is selected from -CI or -Br.
R2 is selected from hydrogen, halogen, -CN, -CF3, -OH, -0(d_4 alkyl), -NH2, -NH(d_4 alkyl), or - (CW alkyl)(CM alkyl). Preferably, R2 is selected from hydrogen, halogen (such as, e.g., -F, -CI, or -Br), -OH, or -NH2. More preferably, R2 is selected from -F, -OH, or -NH2. R3 is selected from -OH, -0(C^ alkyl), -SH, -S(CM alkyl), -NH2l -NH(C^ alkyl), or - N(Ci-4 alkyl)(Ci_4 alkyl). Preferably, R3 is selected from -0(C1-4 alkyl) or -N(C1-4 alkylXd^ alkyl). More preferably, R3 is selected from -0-CH3, -0-CH2-CH3 or ~N(CH3)2, particularly from -O-CH3 or -N(CH3)2.
Alternatively, R2 and R3 jointly form a group -C(halogen)=CH-N(CH3)-, wherein the halogen is preferably selected from -F, -CI, or -Br, and more preferably the halogen is -C!. That is, R2 and R3 form a 5-membered ring together with the carbon atoms which they are attached to, wherein R2 and R3 together are a bivalent group -C(halogen)=CH-N(CH3)- or, preferably, a group -C(CI)=CH-N(CH3)~.
Accordingly, the compound of formula (i) may have the foliowing structure:
halogen
Figure imgf000005_0001
wherein the halogen may, e.g., be -F, -CI, or -Br, and preferably is -CI, and the other groups X and R1 are as defined above.
In a preferred embodiment, R2 is -NH2 and R3 is -O-CH3. In a further preferred embodiment, X is O or N(CH3), R1 is -NH2 or halogen (such as, e.g., -F, -CI, or -Br), R2 is -NH2, and R3 is -O-CH3. Accordingly, a preferred compound of formula (I) is 1-methyl-5-(3-amino-4- methoxyphenyl)-4-(3-chloro-4,5-dimethoxyphenyI)-imidazole (also referred to as "5b" or "compound 5b"). Another preferred compound of formula (I) is 1 -methyI-5-(3-amino-4- methoxyphenyl)-4-(3-bromo-4,5-dimethoxyphenyI)-imidazole (also referred to as "6b" or "compound 6b").
In another preferred embodiment, R2 is -NH2 and R3 is -0-CH2-CH3. In a further preferred embodiment, X is O or N(CH3), R1 is -NH2 or halogen (such as, e.g., -F, -CI, or -Br), Rz is - NH2, and R3 is -0-CH2-CH3. Particularly preferred compounds of formula (!) are, accordingly, 1-methyl-5-(3-amino-4-ethoxyphenyi)-4-(3-chloro-4,5-dimethoxyphenyl)- imidazole (i.e., 1-methyl-5-(3"-amino-4"-ethoxyphenyl)-4-(3'-chloro-4',5,-dimethoxyphenyl)- imidazole; also referred to as "5f or "compound 5f) and 1-methyl-5-(3-amino-4- ethoxyphenyl)-4-(3-bromo-4,5-dimethoxyphenyl)-imidazole (i.e., 1-methyl-5-{3"-amino-4"- ethoxyphenyi)-4-(3'-bromo-4!,5'-dimethoxyphenyi)-imidazole; also referred to as "6F or "compound 6f ).
In another preferred embodiment, R2 is -F and R3 is -0-CH3 or -0-CH2-CH3. In a. further preferred embodiment, X is O or N(CH3), R1 is -NH2 or halogen (such as, e.g., -CI, or -Br), R2 is -F, and R3 is -0-CH3 or -0-CH2-CH3. Accordingly, preferred compounds of formula (I) are 1-methyl-4-(3-chloro-4,5-dimethoxyphenyl)-5-(3-fluoro-4-ethoxypheny!)-imidazole (also referred to as "5g" or "compound 5g") and 1-methyl-4-(3-bromo-4,5-dimethoxyphenyl)~ 5-(3-fiuoro-4-ethoxyphenyl)-imidazole (also referred to as "6g" or "compound "6g").
Further preferred compounds of formula (!) are 1-methyl-4-(3-amino-4,5-dimethoxyphenyi)-5- (N-methyI~3-chloroindol-5-yl)-imidazole (i.e., 1-methyl-4-(3'-amino-4',5'-dimethoxyphenyl)-5- (N-methyl-3"-chloroindol-5"-yl)-imidazole; also referred to as "8e" or "compound 8e"), 1- methyl-4-(3'-chioro-4',5'-dimethoxyphenyl)-5-(N-methyl-3"-chloroindol-5"-yl)-imidazole (also referred to as "5i" or "compound "5i"), and 1-methyl-4-(3'-bromo-4',5!-dimethoxyphenyl)-5-(N- methyl-3"-chloroindol-5"-yl)-imidazole (also referred to as "6Γ or "compound 6i").
Figure imgf000006_0001
The present invention further provides a compound of formula (II)
Figure imgf000006_0002
or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
X2 Is selected from O, S, N(H), or N(C1-4 alkyl). Preferably, X2 is selected from O or N{Ci_4 alkyl). More preferably, X2 is selected from O or N(CH3).
R21 is selected from halogen, -CN, -CF3, -NH2, -NH(C1-4 alkyl), or -N(C1-4 a!kyiXd^ alkyl). Preferably, R21 is selected from halogen (such as, e.g., -F, -CI, or -Br), or -NH2. More preferably, R21 is selected from -CI, -Br, or -NH2. Most preferably, R21 is selected from -CI or -Br.
R22 is Ci_4 aikyi. Preferably, R22 is C2_ aikyl. More preferably, R22 is ethyi.
The group "halogen" comprised in the compound of formula (II) is preferably selected from - F, -CI, or -Br, and more preferably the "halogen" is -CI.
Preferred compounds of formula (II) are 1 -methyl-4-(3-amino-4,5-dimethoxyphenyl)-5-(N- methyl-3-chloroindol-5-yl)-imidazole, l -methyl^-iS'-chloro^'.S'-dimethoxyphenylJ-S-iN- methyl-3"-chloroindol-5,,-yi)-imidazole, and l-meihyi^-iS'-bromo^S'-dimethoxyphenyiy-S- (N-methyl-3"-chloroindoi-5"-y!)-imidazole. A particularly preferred compound of formula (II) is 1-methyl-4-(3'-bromo-4',5,-dimethoxyphenyl)-5-(N-ethyi-3"-chloroindo!-5"-yl)-imidazole (also referred to as "6h" or "compound 6h").
Figure imgf000007_0001
The invention also provides a compound of formula (ill)
Figure imgf000008_0001
(III) or a pharmaceuticaliy acceptable salt, solvate, or prodrug thereof.
X3 is selected from O, S, N(H), or (CM alkyl). Preferab!y, X3 is selected from O or N(C1-4 alkyl). More preferabiy, X3 is selected from O or N(CH3).
R3 is selected from halogen (particularly -CI, -Br or -I), -CN, -CF3, -NH2, - HfC^ alky!), or - (d_ alkyi)(Ci_4 alkyl). Preferably, R3 is selected from -Ci, -Br, -I, or -NH2. More preferably, R31 is selected from -Br, -I, or -NH2. Most preferably, R31 is selected from -Br or -I.
R32 is selected from hydrogen, halogen, -CN, -CF3, -OH, -0(C^ alkyl), -NH2, -NH(C- alky!), or -N(CH a!kyi)(C1_4 alkyl). Preferably, R32 is selected from hydrogen, halogen (such as, e.g., -F, -CI, or -Br), -OH, or -NH2. More preferably, R32 is selected from -F, -OH, or -NH2. Most preferably, R32 is -F.
R33 is selected from -OH, -0(C1-4 alkyl), -SH, -S{d^ alkyl), -NH2, -NH(CM alkyl), or - N(C!_4 alkyt)(C _4 alkyl). Preferably, R33 is selected from -0(01-4 alkyl) or -N(C1_4 alkyI)(C1_4 alkyl). More preferably, R33 is selected from ~0-CH3s ™0-CH2-CH3 or -N(CH3)2. Most preferably, R33 is selected from -0-CH3 or -0-CH2-CH3.
Alternatively, R32 and R33 jointly form a group -C(halogen)=CH-N(C1-4 alkyl)-. The C1-4 alkyl comprised in that group is preferably selected from methyl or ethyl. The halogen comprised in that group is preferably selected from -F, -CI, or -Br, and more preferably the halogen is - CI. That is, R32 and R33 form a 5-membered ring together with the carbon atoms which they are attached to, wherein R32 and R33 together are a bivalent group -C(hatogen)=CH-N(C1_4 alkyl)™. It is particularly preferred that R32 and R33 jointly form a group -C(halogen)=CH- N(CH3)- or a group -C(halogen)=CH-N(CH2CH3)-, wherein in each case the halogen is preferably selected from -F, -CI, or -Br, and more preferably the halogen is -CI.
FT1 is selected from -OH, -0(CW alkyl), -SH, -S(d_4 alkyl), halogen (particularly -CI, -Br or -I), -CN, -CF3, -NH2, -NH(C^ a!kyl), or -N(C^ aikyl)(CM alkyl). Preferably, R34 is selected from -0(C^ alkyl), -CI, -Br, -I, or -NH2. More preferably, R34 is selected from -O- CHs, -0-CH2-CH3l -Br, -I, or -NH2. Most preferably, R34 is selected from -0-CH3, -Br or - I. in a preferred embodiment, R is -F and R is -0-CH3. In a further preferred embodiment, X3 is N(CH3), R31 is -Br or -I, R32 is -F, R33 is -0-CH3, and R34 is -0-CH3, -Br or -I. Particularly preferred compounds of formula (lii) are the following compounds 9a, 9b and 9c:
Figure imgf000009_0001
9a 9b 9c
As used herein, the term "alkyl" refers to a monovalent saturated aliphatic (i.e. non-aromatic) acyclic hydrocarbon group (i.e. a group consisting of carbon atoms and hydrogen atoms) which may be linear or branched and does not comprise any carbon-to-carbon double bond or any carbon-to-carbon triple bond. Accordingly, the term "C^ alky!" refers to methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, tert-butyl, or sec-butyl).
As used herein, the term "halogen" refers to -F, -CI, -Br, or -I, and in particular to -F, -CI, or -Br. The present invention also relates to a pharmaceutical composition comprising a compound of formula (I), (II) or (III) as defined herein or a pharmaceutically acceptable salt, solvate, or prodrug thereof, in combination with a pharmaceuticaliy acceptable excipient. Accordingly, the compounds of formula (I), (II) or (III) are useful as medicaments. The present invention further relates to a compound of formula (I), (II) or (III) as defined herein, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, or a pharmaceutical composition comprising any of the aforementioned entities in combination with a pharmaceutically acceptable excipient, for use in the treatment or prevention of cancer. The use of the above compound for the preparation of a medicament for the treatment or prevention of cancer is also within the scope of the invention.
Furthermore, the invention encompasses a method of treating or preventing cancer, the method comprising the administration of a compound of formula (I), (II) or (III) as defined herein, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, or a pharmaceutical composition comprising any of the aforementioned entities in combination with a pharmaceutically acceptable excipient, to a subject in need of such a treatment or prevention. The cancer to be treated or prevented with the compounds or the pharmaceutical compositions according to the present invention includes, for example, breast (mamma) cancer, genitourinary cancer (such as, e.g., prostate tumor, including a hormone-refractory prostate tumor, or germ cell cancer), lung cancer (such as, e.g., small cell or non-small cell lung tumor), gastrointestinal cancer (such as, e.g., hepatocellular carcinoma, colorectal tumor, colon cancer or gastric cancer), epidermoid cancer (such as, e.g., epidermoid head and/or neck tumor or mouth tumor), melanoma, ovarian cancer, pancreas cancer, neuroblastoma, head and/or neck cancer, bladder cancer, renal cancer, brain cancer, leukemia (such as, e.g., lymphocytic leukemia or myelogenous leukemia), or lymphoma. It is particularly preferred that the cancer to be treated or prevented with the compounds or the pharmaceutical compositions according to the invention is a multidrug-resistant cancer. The cancer to be treated may thus be a multidrug-resistant form of the above described cancers. In a preferred embodiment of the present invention, the cancer to be treated or prevented with the compounds or the pharmaceutical compositions provided herein is resistant against com breta statin A-4 and/or cisplatin. Accordingly, in the present invention the treatment or prevention of combretastatin A-4 (CA-4)-refractory cancer or cisplatin- refractory cancer is particularly envisaged.
The compounds according to the invention have been found to be surprisingly effective in the medical intervention of multidrug-resistant cancer and, in particular, of CA4- refractory cancer, which has been demonstrated in vitro and in vivo, including in a mouse xenograft model (cf. Example 3 and Figures 3, 4 and 5). It has been demonstrated that the new compounds exhibit a distinct cytotoxic, antiangiogenic/antivascular and antimitotic effect. In addition, the imidazole/oxazole bridge in the molecular structure of the new compounds gives rise to a stable cis-configu ration of the C=C-double bond between the arene rings and thus to a high chemical and pharmacological stability. Any isomerization to a trans-stilben with loss of anti-tumor activity is no longer possible. Moreover, their polar substituents render the new compounds very well soluble in water and so directly applicable without employment of a vehicle. These compounds can be administered by injection as solutions in physiological saline solution. Such solutions were shown to be stable for at least one month under ambient conditions without any noticeable loss of activity. In animal studies of a multi-resistant tumor model the new compounds were found anti-tumor active causing a dramatic regression of the tumor xenografts. The strong antivascular effect selectively affecting the tumoral vasculature was unambiguously confirmed by visible hemorrhages of the tumoral tissue. No unwanted side effects were observed when the new compounds were applied at efficacious concentrations. This underscores their high tumor selectivity and their excellent tolerance in vivo. Thus the attachment of special ring substituents at defined positions, as described herein for the compounds of formula (I), (II) and (111), results in enhanced anti-tumor activity, improved pharmacological properties as well as in superior tolerance in vivo and in the potential to overcome chemoresistance of tumors. Furthermore, the compounds of the invention have surprisingly been found to exhibit a synergistic effect resulting from the combination of a high cytotoxicity and a strong antivascular effect selectively affecting the tumoral vasculature, as also demonstrated in the Examples. The nature of the halogen substituent in R in formula (I) is crucial for the selectivity of the compounds according to the invention and the magnitude of their bioactivity. Unlike its chioro congener 5b (structure shown in Example 4), the bromo imidazole 6b was vascular disrupting in tumor xenografts while leaving regular vasculature in chicken embryos (CAM assay) alone. The compounds of the invention are also characterized by an outstanding tumor-selective cytotoxicity and a strong induction of cancer cell apoptosis.
The compounds of the invention, including the compounds of formula (I), further have advantageous properties with respect to solubility in water and chemical stability in solution, as also demonstrated in Example 3 and Figures 1 and 2. For example, the solubility of the compounds 5b, 6b and 8a according to the present invention in water exceeded a concentration of 10 mg/mL Thus, these compounds are even more water-soluble than the potassium salt of combretastatin A-4-phosphate (about 5 mg/mL; Bedford et a!., Bioorg. Med. Chem. Lett. 1996, 6, 157-160). Also for this reason, the compounds of the invention, including the compounds of formula (I), are particularly suitable and effective as medicaments, including as medicaments in the treatment or prevention of cancer. Thus, the compounds of the present invention are particularly advantageous in respect of cytotoxicity, solubility in water, and chemical stability, as has been shown in the appended examples for the compound 5b according to the invention. In particular, compound 5b is highly cytotoxic in vitro and in vivo (likewise in CA-4- and cisplatin-resistant cell Sines), shows an improved water solubility compared with CA-4-phosphate and can not isomerize to inactive frans-isomers iike CA-4. Compound 5b is nevertheless well tolerated and selective in mouse xenografts. The compounds of the present invention can thus be administered without the need of prodrug forms, such as phosphate prodrug forms used for combretastatin-A4. However, in one embodiment prodrugs can be used, if desired. For a person skilled in the field of synthetic chemistry, various ways for the preparation of the compounds of the present invention, including the compounds of formula (I), (II) or (III), will be readily apparent. For example, the compounds of formula (I) can be prepared according to the following general protocol. First, the respective para-toluenesu!fonylmethylisocyanides (Tos iC derivatives) are prepared (Scheme 1 ). Commercially available 5-chloro- or 5-bromovanillin 1a/b were reacted with iodomethane and potassium carbonate to give the veratraldehydes 2a/b which were converted to the tosylmethylformamides 3a/b by reaction with para-toluenesulfinic acid and formamide in the presence of camphorsulfonic acid, !n the same way, 5-nitrovanillin (1c) as obtained by reaction of vanillin with fuming nitric acid in acetic acid was methylated to give veratraldehyde 2c which was converted to formamide 3c, Subsequently, the formamides 3 were dehydrated to the 3-substituted TosM!C-derivatives 4 by treatment with phosphoroxychloride.
Figure imgf000012_0001
Scheme 1 : Synthesis of para-toluenesuifonylmethySisocyanides 4. Reagents and conditions: (i) CH3I, K2C03, TBAi, DMF, 20°C, 24h, 80-90%; (ii) HCONH2, camphorsulfonic acid, para-toluenesulfinic acid, 60°C, 16h, 51-58%; (iii) POCl3, Et3N, D E, -5°C, 3h, 57-74%.
The halo-substituted Tos iC derivatives 4a/b are converted to the imidazoles 5/6 by reaction in dimethoxyethane/ethano! mixtures with aryl aldehydes or the imines generated from the latter (Scheme 2), Selective reduction of the nitro group with Zn/HCI affords the desired amines 5b or 6b, respectively. Treatment of the imidazoles 5a- and 6b-d with 3 M HCl/dioxane yields the respective water-solub!e hydrochlorides.
Figure imgf000013_0001
4a: Hal = CI CI (5), Br (6)
4b: Hal = Br a = R2 = N02, R3 = OCH3
b = R2 = NH2l R3 = OCH3
c = R2 = F, R3 = OCH3
R2 = H, R3 = N(CH3)2
Scheme 2: Synthesis of N-methylimidazole-bridged combretastatin A4-analogs. Reagents and conditions: (i) ArCHO, CH3NH2 (33% in ethanol), CH3C02H, ethanol, reflux, 2h; then 4a/b, K2C03, dimethoxyethane/ethanol, reflux, 6h, 52-99%; (ii)
Zn, HCI, THF, 20°C, 10 min, 40-91 %.
The nitro-substituted compounds 7a-e are prepared similarly to the imidazoles 5/6 from the nitro-TosMIC derivative 4c and the corresponding aldehydes and imines (Scheme 3). The amines 8a-e are obtained by Pd-catalyzed transfer hydrogenation. The 3-chloroindole 7e is reduced with Zn/HCI to amine 8e. The compounds 8 can further be converted to water-solubie hydrochlorides.
Figure imgf000014_0001
4c 7 8a = X = O, R2 = NH2l R3 = OCH3
8b = X = O, R2 = H, R3 = N{CH3)2 8c = X = NCH3, R2 - OH, R3 = OCH3 8d = X = NCH3, R2 = F, R3 = OCH3 8e = X = NCH3, R2— R3 = CCl-CH-N(CH3
Scheme 3: Synthesis of water-soluble amino substituted oxazoles and N- methylimidazoles 8. Reagents and conditions: (i) ArCHO, K2C03, dimethoxyethane/CH3OH, reflux, 2h, 42-86% (for X = O); ArCHO, CH3NH2 (33% in ethanol), CH3C02H, ethanol, reflux, 2h; then 4c, K2C03 dimethoxyethane/ethanol, reflux, 6h, 64-74% (for X = NCH3); (ii) HC02NH4, Pd/C (5%), CH3OH, reflux, 2h, 67-84% (for 8a-d); Zn, HCI, THF, 20°C, 10 min, 64% (for 8e). The compounds 5e-g and 6e-g are prepared as follows (Scheme 4). 4-Ethoxy- 3-nitro/fluorobenza!dehyde is treated with MeNH2 to give imine intermediates, which are reacted with the TosMIC reagents 4a/b under basic conditions to give the N-methyl imidazoles 5e,g and 6e,g. The preparation of the amines 5f and 6f is accomplished by reduction of 5e/6e with Zn/HCI in THF. These compounds can be converted into the hydrochloride salts, e.g., by treatment with 3 M HCI/dioxane.
Figure imgf000014_0002
4 .... i— 5e: R CI, R2 = N02
a: R = CI (,,)^ 5f: R^ = CI, R2 = NH2 x 2HCI
b: R = Br 5g: R1 CI, R2 = F x HCI
...,r- 6e: R Br, R2 = N02
Br, R2 = NH2 X 2HCI
6g: R1 Br, R2 = F x HCI
Scheme 4: Synthesis of compounds 5e-g and 6e-g. Reagents and conditions: (i) subst.
ArCHO, MeNH2, AcOH, EtOH, reflux, 2 h, then 4a/b, K2C03, EtOH, reflux, 3h; (ii) Zn, HCI, THF, r.t., 15 min, then 3M HCI/dioxane, DC , r.t., 15 min.
Furthermore, the compounds of formula (I) can also be prepared in analogy to the synthesis routes described in Wang et a!., J. Med. Chem. 2002, 45, 1697-171 1.
The compounds of formula (II) or (111) can be prepared in accordance with or in anaiogy to the syntheses described above and/or in the Examples, The compounds of formula (ill), in particular, can also be prepared according to the fo!iowing general protocol. The compounds 9a-c are prepared as fo!lows (Scheme 5). 3-Bromo-4,5-dimethoxy- benzaldehyde, 3,5-dibromo-4-methoxybenzaldehyde, and 3,5-diiodo-4-methoxybenz- aldehyde, respectively, are treated with eNH2 to give imine intermediates, which are reacted with the TosMIC reagent 4d under basic conditions to give the N-methyl imidazoles 9a-c. These compounds can be converted to the hydrochloride salts, e.g., by treatment with 3 M HCI/dioxane.
Figure imgf000015_0001
4d 9a: R1 = Br, R2 = OMe
9b: R1, R2 = 8r
9c: R1, R2 = I
Scheme 5: Synthesis of compounds 9a-c. Reagents and conditions: (i) subst. ArCHO,
MeNH2) AcOH, EtOH, reflux, 2 h, then 4d, K2C03, EtOH, reflux, 3h.
The scope of the invention embraces all pharmaceutically acceptable salt forms of the compounds of the present invention, in particular the compounds of formula (I), (II) or (III), which may be formed, e.g., by protonatton of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of a carboxylic acid group with a physio!ogically acceptable cation as they are well known in the art. Exemplary base addition salts comprise, for example, alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; ammonium salts; aliphatic amine salts such as trimethyiamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamtne, procaine salts, meglumine salts, diethanol amine salts or ethylenediamine salts; aralkyl amine salts such as Ν,Ν-dibenzy!ethylenediamine salts, benetamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts or isoquinoiine salts; quaternary ammonium salts such as tetramethyiammonium salts, tetraethylammonium salts, benzyltrimethylarnmonium salts, benzyltriethylammonium salts, benzyltributyiammonium salts, methyltrioctyiammonium salts or tetrabutylammonium salts; and basic amino acid salts such as arginine salts or lysine salts. Exemplary acid addition salts comprise, for example, mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts, nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dthydrogenphosphate salts), carbonate salts, hydrogencarbonate salts or perchlorate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, undecanoate, lactate, maleate, oxalate, fumarate, tartrate, maiate, citrate, nicotinate, benzoate, salicylate or ascorbate salts; sulfonate salts such as methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-toluenesulfonate (tosy!ate), 2-naphthalenesulfonate, 3-phenylsulfonate, or camphorsulfonate salts; and acidic amino acid salts such as aspartate or glutamate salts.
Moreover, the scope of the invention embraces solid forms of the compounds of formula (I), (II) or (111) in any soivated form, including e.g. solvates with water, for example hydrates, or with organic solvents such as, e.g., methanol, ethanol or acetonitriie, i.e. as a methanolate, ethanolate or acetonitrilate, respectively; or in the form of any polymorph.
Furthermore, the formulas in the present application are intended ΐο cover all possible stereoisomers, including enantiomers and diastereomers, of the indicated compounds.
Thus, all stereoisomers of the compounds of formula (I), (II) or (111) are contemplated as part of the present invention, either in admixture or in pure or substantially pure form. The scope of the compounds according to the invention embraces all the possible stereoisomers and their mixtures. It particularly embraces the racemic forms and the isolated optical isomers. The racemic forms can be resolved by physical methods, such as, e.g., fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chirai column chromatography. The individual optical isomers can be obtained from the racemates using conventional methods, such as, e.g., salt formation with an optically active acid followed by crystallization.
Pharmaceutically acceptable prodrugs of the compounds of formula (!), (II) or (III) are derivatives which have chemically or metabolicai!y cleavable groups and become, by soivolysis or under physiological conditions, the compounds of the present invention which are pharmaceutically active in vivo. Prodrugs of the compounds of formula (I), (II) or (III) may be formed in a conventional manner with a functional group of the compounds such as with an amino or a hydroxy! group. The prodrug derivative form often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgaard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). When a compound employed in the present invention has a hydroxy! group, an acyloxy derivative prepared by reacting the hydroxyl group with a suitable acylhalide or a suitable acid anhydride is exemplified as a prodrug. An especially preferred acyloxy derivative as a prodrug is - OC(=0)-CH3, -OC(=0)-C2H5, -OC(=0)-(tert-Bu), -OC(=0)-C15H31, -OC(=0)-(m-COONa- Ph), -OC(=0)-CH2CH2COONa! -0(C=0)-CH(NH2)CH3 or -OC(=0)-CH2-N(CH3)2. When a compound employed in the present invention has an amino group, an amide derivative prepared by reacting the amino group with a suitable acid halide or a suitable mixed anhydride is exemplified as a prodrug. An especially preferred amide derivative as a prodrug is -NHC(=OHCH2)2OCH3 or -NHC(=0)-CH(NH2)CH3. Accordingly, the compound of formula (I) may be used as a prodrug, wherein the prodrug is a compound of formula (!), in which one or more amino groups, if present, are in the form of an amide, such as e.g. in the form of -NHC(=0)-(CH2)2OCH3 or -NHC(=0)-CH(NH2)CH3, or in which one or more hydroxyl groups, if present, are in the form of an acyloxy, such as e.g. in the form of - OC(=0)-CH3! -OC(=0)-C2H5, -OC(=0)-(tert-Bu),
-OC(=0)-C1sH31, -OC(=0)-(m-COONa-Ph), ~OC(=0)-CH2CH2COONa, ~0(C=0)~ CH(NH2)CH3 or -OC(=0)-CH2-N(CH3)2, or in the form of a phosphate, i.e. in the form of -O- P(=0)(0~)2, or a phosphate salt (such as, e.g., the base addition salts described herein above). Thus, if R2 and/or R3 in the compound of formula (l) is -OH (i.e., hydroxyl), then a corresponding prodrug may be the compound of formula (I), in which the hydroxyl group which is R2 (if applicable) and/or the hydroxy! group which is R3 (if applicable) is in the form of a phosphate (i.e. in the form of -0-P(=0)(0~)2), a phosphate salt, or an acyloxy, preferably in the form of a phosphate or a phosphate salt (e.g., with sodium, potassium, or any of the cations referred to in respect of the base addition salts described herein above). A preferred prodrug of the compounds of the present invention is a compound of formula (I), in which R2 is -0-P(=0)(OH)2 or a salt thereof (e.g., a disodium or a dipotassium salt).
The compounds described herein may be administered as compounds per se or may be formulated as medicaments. The medicaments/pharmaceutical compositions may optionally comprise one or more pharmaceutically acceptable excipients, such as carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, or solubility enhancers. In particular, the pharmaceutical compositions may comprise one or more solubility enhancers, such as, e.g., poly(ethy!ene glycol), including poly{ethylene glycol) having a molecular weight in the range of about 200 to about 5,000 Da, ethylene glycol, propylene glycol, non-ionic surfactants, tyloxapol, polysorbate 80, macrogol-15-hydroxystearate, phospholipids, lecithin, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, cyc!odextrins, hydroxyethyl-p-cyclodextrin, hydroxypropyl-β- cyclodextrin, hydroxyethyi-Y-cycSodextrin, hydroxypropyl-y-cyclodextrin, dihydroxypropyl-β- cyclodextrin, glucosyl-a-cyclodextrin, glucosyl^-cyclodextrin, diglucosyl- -cyclodextrin, maltosyl-a-cyclodextrin, maltosyl- -cyclodextrin, maltosyl-y-cyciodextrin, maltotriosyl-β- cyclodextrin, maltotriosyl-y-cyclodextrin, dimaitosyl-p-cyclodextrin, methyi^-cyclodextrin, carboxyalkyi thioethers, hydroxypropyi methylceliulose, hydroxypropyicelluiose, polyvinylpyrrolidone, vinyl acetate copolymers, vinyl pyrroiidone, sodium lauryl sulfate, dioctyl sodium suSfosuccinate, or any combination thereof.
The pharmaceutical compositions can be formulated by techniques known to the person skilled in the art, such as the techniques published in Remington's Pharmaceutical Sciences, 20th Edition. The pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as intramuscular, intravenous, intraperitoneal, subcutaneous, intradermal, intraarterial, rectal, nasal, topical, aerosol or vaginal administration. Dosage forms for oral administration include coated and uncoated tablets, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets. Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions and powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration. Dosage forms for rectal and vaginal administration include suppositories and ovula. Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler. Dosage forms for topical administration include creams, gels, ointments, salves, patches and transdermal delivery systems.
The compounds of formula (I), (II) or (111) or the above described pharmaceutical compositions comprising a compound of formula (I), (I!) or (111) may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to one or more of: oral (e.g. as a tablet, capsule, or as an ingestible solution), topical (e.g., transdermal, intranasal, ocular, buccal, and sublingual), parenteral (e. g., using injection techniques or infusion techniques, and including, for example, by injection, e.g. subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, or intrastemal by, e.g., implant of a depot, for example, subcutaneously or intramuscularly), pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g. through mouth or nose), gastrointestinal, intrauterine, intraocular, subcutaneous, ophthalmic (including intravitreal or intracamerai), rectal, and vaginal. In particular, the compounds or the pharmaceutical compositions may be administered oraliy or by inhalation. It is particularly preferred that the compounds of the present invention are administered by pulmonary administration, in particular by inhalation, in the form of dry powder formulations as described e.g. in more detail herein below.
If the compounds or pharmaceutical compositions are administered parenterally, then examples of such administration include one or more of: intravenously, intraarteriai!y, intraperitoneal, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the compounds pharmaceutical compositions, and/or by using infusion techniques. For parenteral administration, the compounds are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. The compounds or pharmaceutical compositions can also be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications. The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disint eg rants such as starch (preferably com, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylceilulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, miik sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanot, propylene glycol and glycerin, and combinations thereof.
Alternatively, the compounds or pharmaceutical compositions can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The compounds of the present invention may also be derma!ly or transdermal administered, for example, by the use of a skin patch.
The compounds or pharmaceutical compositions may also be administered by the pulmonary route, rectal routes, or the ocular route. For ophthalmic use, they can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
It is furthermore envisaged to prepare dry powder formulations of the compounds of the present invention for pulmonary administration. The dry powder formulations of the compounds of the present invention may be delivered using any suitable dry powder inhaler (DPI), i.e., an inhaler device that utilizes the patient's inhaled breath as a vehicle to transport the dry powder drug to the lungs.
For topical application to the skin, the compounds or pharmaceutical compositions can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propyiene glycol, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, 2-octyldodecanol, benzyl alcohol and water.
Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular individual subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual subject undergoing therapy.
A proposed, yet non-limiting dose of the compounds of the invention, including the compounds of formula (I), (II) or (III), for administration to a human (of approximateiy 70 kg body weight) may be 0.1 pg to 10 g, preferably 0.1 mg to 1 g, and more preferably about 200 mg or more (e.g., 200 to 300 mg or 200 to 500 mg), of the active ingredient per unit dose. The unit dose may be administered, for example, 1 to 4 times per week. The dose will depend on the route of administration. It will be appreciated that it may be necessary to make routine variations to the dosage depending on the age and weight of the patient/subject as well as the severity of the condition to be treated. The precise dose and route of administration will ultimately be at the discretion of the attendant physician or veterinarian. in one embodiment, the compounds according to the present invention can be used in combination with other therapeutic agents. When the compound is used in combination with a second therapeutic agent active against the same disease, the dose of each compound may differ from that when the compound is used alone. The combination of a compound of this invention with (an) other drug(s) may comprise the administration of the drug(s) with the compound of the invention. Such an administration may comprise simultaneous/concomitant administration. However, sequentiai/separate administration is also envisaged.
Preferably, the second therapeutic agent to be administered in combination with the compound of the present invention is an anticancer drug. The anticancer drug to be administered in combination with the compound of the invention may be: a tumor angiogenesis inhibitor (for example, a protease inhibitor, an epidermal growth factor receptor kinase inhibitor, or a vascular endothelial growth factor receptor kinase inhibitor); a cytotoxic drug (for example, an antimetabolite, such as purine and pyrimidine analogue antimetabolites); an antimitotic agent (for example, a microtubule stabilizing drug or an antimitotic alkaloid); a platinum coordination complex; an anti-tumor antibiotic; an alkylating agent (for example, a nitrogen mustard or a nitrosourea); an endocrine agent (for example, an adrenocorticosteroid, an androgen, an anti-androgen, an estrogen, an anti-esirogen, an aromatase inhibitor, a gonadotropin-releasing hormone agonist, or a somatostatin analogue); or a compound that targets an enzyme or receptor that is overexpressed and/or otherwise involved in a specific metabolic pathway that is misregulated in the tumor ceil (for example, ATP and OTP phosphodiesterase inhibitors, histone deacetylase inhibitors, protein kinase inhibitors (such as serine, threonine and tyrosine kinase inhibitors (for example, Abelson protein tyrosine kinase)) and the various growth factors, their receptors and kinase inhibitors therefor (such as epidermal growth factor receptor kinase inhibitors, vascular endothelial growth factor receptor kinase inhibitors, fibroblast growth factor inhibitors, insulin-like growth factor receptor inhibitors and platelet-derived growth factor receptor kinase inhibitors)); methionine; aminopeptidase inhibitors; proteasome inhibitors; cyclooxygenase inhibitors (for example, cyclooxygenase-1 or cyclooxygenase-2 inhibitors); or topoisomerase inhibitors (for example, topoisomerase I inhibitors or topoisomerase II inhibitors).
An alkylating agent which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a nitrogen mustard (such as cyclophosphamide, mechiorethamine (chlormethine), uramustine, melphalan, chiorambucil, ifosfamide, bendamustine, or trofosfamide), a nitrosourea (such as carmustine, streptozocin, fotemustine, lomustine, nimustine, prednimusiine, ranimustine, or semustine), an aikyi sulfonate (such as busulfan, mannosulfan, or treosulfan), an aziridine (such as hexamethyimelamine (altretamine), triethyienemelamine, ThioTEPA (Ν,Ν'Ν'- triethylenethiophosphoramide), carboquone, or triaziquone), a hydrazine (such as procarbazine), a triazene (such as dacarbazine), or an imidazotetrazines (such as temozolomide).
A piatinum coordination compiex which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, or triplatin tetranitrate.
A cytotoxic drug which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, an antimetabolite, inciuding folic acid analogue antimetabolites (such as aminopterin, methotrexate, pemetrexed, or raltitrexed), purine analogue antimetabolites (such as ctadnbine, clofarabine, fludarabine, 6-mercaptopurine (including its prodrug form azathioprine), pentostatin, or 6-thioguanine), and pyrimidine analogue antimetabolites (such as cytarabine, decitabine, 5-fluorouraciS (including its prodrug forms capecitabine and tegafur), floxuridine, gemcitabine, enocitabine, or sapacitabine).
An antimitotic agent which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a taxane (such as docetaxei, iarotaxel, ortataxel, paclitaxel/taxoi, or tesetaxel), a Vinca alkaloid (such as vinblastine, vincristine, vinflunine, vindesine, or vinore!bine), an epothilone (such as epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, or epothilone F) or an epothilone B analogue (such as ixabepilone/azaepothilone B). An anti-tumor antibiotic which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, an anthracycline (such as aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, or zorubicin), an anthracenedione (such as mitoxantrone, or pixantrone) or an anti-tumor antibiotic isolated from Streptomyces (such as actinomycin (including actinomycin D), bleomycin, mitomycin (including mitomycin C), or plicamycin).
A tyrosine kinase inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, axitinib, bosutinib, cediranib, dasatinib, eriotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxanib, sorafenib, sunitinib, or vandetanib.
A topoisomerase-inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a topoisomerase I inhibitor (such as irinotecan, topotecan, camptothecin, be!otecan, rubitecan, or lamellarin D) or a topoisomerase II inhibitor (such as amsacrine, etoposide, etoposide phosphate, teniposide, or doxorubicin).
Further anticancer drugs may be used in combination with a compound of the present invention. The anticancer drugs may comprise biological or chemical moiecules, like TNF- related apoptosis-inducing ligand (TRAIL), tamoxifen, amsacrine, bexarotene, estramustine, irofulven, trabectedin, cetuximab, panitumumab, tositumomab, alemtuzumab, bevacizumab, edrecolomab, gemtuzurnab, alvocidib, seiiciclib, aminolevulinic acid, methyl aminolevulinate, efaproxiral, porfimer sodium, talaporfin, temoporfin, verteporfin, alitretinoin, tretinoin, anagrelide, arsenic trioxide, atrasentan, bortezomib, carmofur, celecoxib, demecolcine, elesclomol, elsamitrucin, etogiucid, lonidamine, lucanthone, masoprocol, mitobronitol, mitoguazone, mitotane, ob!imersen, omacetaxine, sitimagene, ceradenovec, tegafur, testoiactone, tiazofurine, tipifarnib, and vorinostat. Also biological drugs, like antibodies, antibody fragments, antibody constructs (for example, single-chain constructs), and/or modified antibodies (like CDR-grafted antibodies, humanized antibodies, "full humanized" antibodies, etc.) directed against cancer or tumor markers/factors/cytokines involved in proliferative diseases can be employed in co-therapy approaches with the compounds of the invention. Examples of such biological molecules are anti-HER2 antibodies (e.g. trastuzumab, Herceptin®), anti-CD20 antibodies (e.g. Rituximab, Rituxan®, abThera®, Reditux®), anti-CD19/CD3 constructs (see, e.g., EP-A-1 071 752) and anti-TNF antibodies (see, e.g., Taylor et ai., Antibody therapy for rheumatoid arthritis, Curr Opin Pharmacol, 2003, 3(3), 323-328). Further antibodies, antibody fragments, antibody constructs and/or modified antibodies to be used in co-therapy approaches with the compounds of the invention can be found in: Taylor et a!., Antibody therapy for rheumatoid arthritis, Curr Opin Pharmacol, 2003, 3(3), 323-328; or Roxana et al., Maedica, 2006, 1 (1 ), 63-65.
The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formuiation. The individual components of such combinations may be administered either sequentially or simultaneously/concomitantly in separate or combined pharmaceutical formulations by any convenient route. When administration is sequential, either the present compound or the second therapeutic agent may be administered first. When administration is simultaneous, the combination may be administered either in the same or different pharmaceutical composition. When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation. When formulated separately they may be provided in any convenient formulation, conveniently in such manner as are known for such compounds in the art.
In another embodiment, the compounds of the present invention are administered in combination with physical therapy, such as radiotherapy. Radiotherapy may commence before, after, or simultaneously with administration of the compounds. For example, radiotherapy may commence 1 to 10 minutes, 1 to 10 hours or 24 to 72 hours after administration of the compounds. Yet, these time frames are not to be construed as limiting. The subject is exposed to radiation, preferably gamma radiation, whereby the radiation may be provided in a single dose or in multiple doses that are administered over several hours, days and/or weeks. Gamma radiation may be delivered according to standard radiotherapeutic protocols using standard dosages and regimens. Without being bound by theory, the compounds of the present invention may be used to render cells, in particular undesired protiferative/hyperpro!iferative ceils like cancer or tumor cells, more susceptible to such a physical therapy, e.g. radiotherapy.
Accordingly, the present invention relates to a compounds of formula (I), (il) or (III) or a pharmaceutically acceptable salt, solvate, or prodrug thereof, or a pharmaceutical composition comprising any of the aforementioned entities in combination with a pharmaceutically acceptable excipient, for use in the treatment or prevention of cancer, whereby the compound or the pharmaceutical composition is to be administered in combination with an anti-proliferative drug, an anticancer drug, a cytostatic drug, a cytotoxic drug and/or radiotherapy.
Furthermore, it is particularly envisaged to use the combination therapy as described herein above for the treatment or prevention of multidrug-resistant cancer, including the specific multidrug-resistant cancers described above.
The subject or patient, such as the subject in need of treatment or prevention, may be an animal, a vertebrate animal, a mammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), a murine (e.g. a mouse), a canine (e.g. a dog), a feline (e.g. a cat), an equine (e.g. a horse), a primate, a simian (e.g. a monkey or ape), a monkey (e.g. a marmoset, a baboon), an ape (e. g. gorilla, chimpanzee, orangutang, gibbon), or a human. The meaning of the terms "animal", "mammal", etc. is well known in the art and can, for example, be deduced from Wehner und Gehring (1995; Thieme Verlag). In the context of this invention, it is particularly envisaged that animals are to be treated which are economically, agronomically or scientifically important. Scientifically important organisms include, but are not limited to, mice, rats, rabbits, fruit flies like Drosophila melagonaster and nematodes like Caenorhabditis elegans. Non-limiting examples of agronomically important animals are sheep, cattle and pig, whiie, for example, cats and dogs may be considered as economically important animals. Preferably, the subject/patient is a mammal. More preferably, the subject/patient is a human.
The term "treatment of a disorder or disease" as used herein, e.g., in the case of the treatment of cancer, is well known in the art. "Treatment of a disorder or disease" implies that a disorder or disease has been diagnosed in a patient/subject. A patient/subject suspected of suffering from a disorder or disease typically shows specific clinical and/or pathological symptoms which a skilled person can easily attribute to a specific pathological condition (i.e. make a diagnosis of a disorder or disease). "Treatment of a disorder or disease" may, for example, !ead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only). "Treatment of a disorder or disease" may also lead to a partial response (e.g., ameiioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease. "Amelioration" of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse, it is to be understood that a subject/patient may experience a broad range of responses to a treatment (e.g., the exemplary responses as described herein above).
Treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment {including symptomatic relief).
Also the term "prevention of a disorder or disease" as used herein, e.g., in the case of the prevention of cancer, is well known in the art. For example, a patient/subject suspected of being prone to suffer from a disorder or disease as defined herein may, in particular, benefit from a prevention of the disorder or disease. The subject/patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition. Such a predisposition can be determined by standard assays, using, for example, genetic markers or phenotypic indicators. It is to be understood that a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in the patient/subject (for example, the patient/subject does not show any clinical or pathological symptoms). Thus, the term "prevention" comprises the use of compounds of the present invention before any clinical and/or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.
In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
The invention is also illustrated by the following illustrative figures. The appended figures show:
Figure 1: Inhibition of tubulin polymerization by 5b (freshly prepared solution vs solution having been stored for one month). Compound 5b effectively inhibits the polymerization of tubulin. No loss of activity is observed over time. (Test: Tubulin-Polymerization assay). Figure 2: Cytotoxic activity of 5b (freshly prepared solution vs solution having been stored for one month). The persistence of efficacy is proof of the chemical stability of 5b when dissolved (Test: SRB-cytotoxicity assay). Figure 3: Comparison of the in vitro cytotoxicities of 5b and cispiatin (A) or of 5b, 6b and cisplatin (B). Treatment with 5b or 6b results in a breach of the multi-drug resistance of 141 1 HP cells (Test: SRB-cytotoxicity assay).
Figure 4: In vivo anti-tumor activity of compounds 5b and 6b in mouse xenografts of multi- resistant germ cell tumor eel! Sine 141 1 HP. The tumor response following sing!e-dose (A) and dual-dose (B) applications of test compounds, and the tumor response to repeated applications (C) are shown. Arrows indicate the administration of test compounds.
Figure 5: Vascular disrupting effect of 5b. The distinct vascular disrupting effect selectively affecting the tumoral vasculature is apparent from hemorrhages leading to red-blue to brown coloring of the entire tumor (A: before treatment; B: 24 hours after start of treatment) (Test: subcutaneous tumor xenografts in a nude mouse model; mice were anesthesized for the imaging).
Figure 6: Cell growth inhibition by reference compound 25f and the compounds 5b, 6b, 8a and 8e or 5c, 5d, 6c, 6d, 8b, 8c and 8d according to the invention (structures are indicated in Example 2 or 4) at various concentrations {■: 100 μΜ; A : 1 μΜ; V : 0.01 μΜ; 0.001 μΜ) in ceils of human 518A2 melanoma, HL-60 leukemia, HT-29 colon adenocarcinoma, KB- V1/Vbl cervix carcinoma and MCF-7/Topo breast adenocarcinoma (A) or in cells of human 5 8A2 melanoma, HL-60 leukemia and HT-29 colon adenocarcinoma (B) upon incubation for 24 - 72 h (x-axis). Y-axis shows number of viable cells relative to untreated controls (1 ) as ascertained by the MTT assay.
Figure 7: Ceil growth inhibition by combretastatin A-4 at various concentrations (■: 100 μΜ; A : 1 μΜ; #: 0.01 μΜ; ♦: 0.001 μΜ) in cells of human HT-29 colon adenocarcinoma, incubation for 24-72 h (x-axis). Y-axis shows number of viable cells relative to untreated controls as ascertained by the MTT assay.
Figure 8: Microscopic images of HL-60 cells tested in TUNEL assays after 16 h incubation with 10 μΜ of the compound 5b, 6b or 8a. The brightfie!d pictures (left) show all the cells in the focus, the pictures of the green fluorescent channel (right) just the apoptotic cells, represented as bright dots.
Figure 9: Chicken embryos with surrounding blood vessels immediately after adding the test compounds CA-4 or 5b (left), after one day (middle) and after three days (right) are shown. The top row shows a negative controi. Pictures are representative of at least two independent runs.
Figure 10: In vivo anti-tumor activity of compound 6b in mouse xenograft of multi-resistant germ cell tumor cell line 1411 HP. The tumor response following dual-dose application is shown. Arrows indicate the administration of test compound.
Figure 11 : Vascular disrupting effect of compounds 5b, 6b, 5f and 6f in A2780 ovarian carcinoma xenograft tumors (A) and 1411 HP germ cell tumor xenografts (B). The distinct vascular disrupting effect selectively affecting the tumoral vasculature is apparent from hemorrhages leading to red-blue to brown coloring of the entire tumor (day 0: before treatment; day 1 : 24 hours after start of treatment) (Test: subcutaneous tumor xenografts in a nude mouse model; mice were anesthesized for the imaging). Figure 12: Vascular disrupting effect in 1411 HP germ cell tumor xenograft after oral administration of 6b. The distinct vascular disrupting effect selectively affecting the tumoral vasculature is apparent from hemorrhages leading to red-blue to brown coloring of the entire tumor (day 0: before treatment; day 1 : 24 hours after start of treaiment) (Test: subcutaneous tumor xenografts in a nude mouse model; mice were anesthesized for the imaging).
Figure 13: Comparison of the in vitro cytotoxicities of compounds 5b, 6b, 5f and 6f compared to 25f in H12.1 germ cell tumor (A), 141 1 HP germ cell tumor (B), A2780 ovarian carcinoma (C), HT29 colon carcinoma (D), DLD1 colon carcinoma (E) and HCT8 colon carcinoma (F) cell lines (Test: SRB-cytotoxtcity assay).
Figure 14: Tube formation assay with HUVEC cells after treaiment with compound 8a (A), 6b (B) or 5b (C) at concentrations of 7.72 ng/mL or control (methanol) (D).
Figure 15: High-content analysis of compound 6b (denoted as "Brimamin (Schobert)") and compound 8a (denoted as "Amoxamin_2 (Schobert)") in PtK-2 cells.
The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention. EXAMPLES
General Remarks Melting points were recorded on a GALLENKAMP apparatus and are uncorrected. I R spectra were recorded on a PERKIN-ELMER Spectrum One FT-IR spectrophotometer with ATR sampling unit. Nuclear magnetic resonance (NMR) spectra were recorded under conditions as indicated on a BRUKER Avance 300 spectrometer. Chemical shifts are given in parts per miilion (<5) downfie!d from tetrameihyi silane as internal standard for 1 H and 13C. Mass spectra were recorded using a VARiAN MAT 31 1 A (Ei), Microanalyses were carried out with a PERKINELMER 2400 CHN elementai analyser. All tested compounds are > 95% pure by elemental anaiysis. For chromatography MERCK silica gel 60 (230-400 mesh) was used. All starting compounds were purchased from ALDRICH and used without further purification.
Example 1 : Synthesis of 1 -methyl-5-(3-amino-4-methoxyphenyi)-4-(3-chioro-4,5- dimethoxyphenyl)-imidazole bis(hydrochloride) (5b x 2 HCI)
1 ) N-t(Toluene-4-sulfonyl)-(3-chloro-4,5-dimethoxyphenyl)methyl]formamide 3a
5-Chloroveratraldehyde (5.7 g, 23.4 mmol), para-toluenesulfinic acid (3.0 g, 19.3 mmol) and camphorsuifonic acid (1 10 mg, 0.47 mmol) were treated with formamide (10 mL). Upon heating to 65 °C the reaction mixture turned into a solution and after two hours the product began to precipitate. After stirring for 16 h the precipitate was filtered, washed with methanol and dried in vacuum.
Yield: 4.57 g (1 1.92 mmol, 51 %); colorless solid of mp 157-158 °C; vmax (ATR)/cm~1; 3190, 3107, 2947, 1690, 1593, 1576, 1484, 1470, 1423, 1403, 1308, 1283, 1250, 1215, 1 143, 1 121 , 1078, 1053, 999, 860, 822, 788, 769, 659, 689; H-NMR (300 MHz, DMSO-tf6): δ 2.41 (3 H, s, CH3), 3.76 (3 H, s, O CH3), 3.79 (3 H, s, O CH3), 6.45 (1 H, d, 3 J 10.7 Hz, CH), 7.24 (1 H, d, 4 J 1 .9 Hz, 2-H), 7.30 (1 H, d, 4J 1 .9 Hz, 6-H), 7.43 (1 H, d, 3 J 8.4 Hz, Tosyl-3-H/5-H), 7.72 (2 H, d, 3 J 8.4 Hz, Tosyl-2-H/6-H), 7.98 (1 H, s, HCONH), 9.74 (1 H, d, 3 J 10.7 Hz, NH); 13C-NMR. (75.5 MHz, DMSO-rf6): δ 21 .1 (CH3), 56.3 (5-OCH3), 60.3 (4-OCH3), 69.5 (CH), 1 13.5 (C-6), 122.4 (C-2), 126.7 (C-3), 127.1 (C- ), 129.2 (Tosyl-C-3/C-5), 129.6 (Tosyl-C- 2/C-6), 133.2 (Tosyi-C-1 ), 144.9 (C-4), 145.4 (Tosyl-C-4), 153.1 (C-5), 160.2 (CO); m/z (%) 382 (4), 278 (6), 227 (89), 192 (76), 156 (57), 1 13 (55), 91 (100), 77 (67), 63 (92). 2) 3-Ch!oro-4,5-dimethoxyphenyl(tosyl)methylisocyanide 4a
Compound 3a (4.57 g, 1 1.92 mmol) was suspended in dry dimethoxyethane (100 mL) and cooled to -10°C. POCi3 (3.4 mL, 36.1 mmol) was added and a mixture of triethylamine (8.3 mL, 59.5 mmol) in dimethoxyethane (10 mL) was dropped slowly to the reaction mixture. After stirring for 2 h at -5 °C, the reaction mixture was poured into ice water. The aqueous phase was extracted with ethyl acetate, the organic phase was washed with saturated aqueous NaHC03 and brine, dried over Na2S04! filtered and concentrated in vacuum. Upon refrigeration (4°C) over night a yellow solid crystallized, which was collected and dried in vacuum.
Yield: 2.48 g (6.79 mmol, 57%); yellow solid of mp 115 °C;, vmax (ATR)/cm"1: 2920, 2136, 1593, 1577, 1492, 1452, 1423, 1325, 1294, 1276, 1238, 1 199, 1 137, 1082, 1053, 1002, 862, 826, 759, 705, 683; 1H-NMR (300 MHz, CDCl3): δ 2.45 (3 H, s, CH3), 3.79 (3 H, s, 5-OCH3), 3.87 (3 H, s, 4-OCH3), 5.49 (1 H, s, CH), 6.76 (1 H, d, 4J 2.1 Hz, 6-H), 6.88 (1 H, d, J 2,1 Hz, 2-H), 7.34 (2 H, d, 3J 8.5 Hz, Tosy!-3-H/5~H), 7.64 (2 H, d, 3J 8.5 Hz, Tosyl-2-H/6-H); 13C- NMR (75.5 MHz, CDCI3): δ 21 .7 (CH3), 56.2 (5-OCH3), 60.8 (4-OCH3), 75.6 (CH), 1 10.8 (C- 6), 122.2 (C-2), 122.6 (C-1 ), 128.6 (C-3), 129.9 (Tosyl-C-3/C-5), 130.0 (Tosyl-C-4), 130.4 (Tosyl-C-2/C-6), 146.9 (Tosyl-C-1 ), 147.4 (C-4), 153.8 (C-5), 166.6 (CN); m/z (%) 365 (2) [M+], 278 (7), 246 (10), 210 (100), 155 (23), 91 (54), 66 (20).
3) 1-lvlethyl-4-(3-chloro-4,5-dimethoxyphenyl)-5-(4-methoxy-3-nitrophenyl)imidazole 5a.
A mixture of 4-methoxy-3-nitrobenzaldehyde (76 mg, 0.42 mmol) and 33% MeNH2/ethanol (260 pL, 2.10 mmol) in ethanol (15 mL) was treated with acetic acid (150 pL) and refluxed for 2 h. After cooling to room temperature, compound 4a (153 mg, 0.42 mmol) dissolved in dimethoxyethane (10 mL) and K2C03 (500 mg, 3.62 mmol) was added and the reaction mixture was refluxed for another 3 h. The solvent was evaporated, the residue diluted with ethyl acetate, washed with water and brine, dried over Na2S0 , filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60).
Yield: 1 10 mg (0.26 mmol, 62%); yellow oil; Rf = 0.24 (ethyl acetate/m ethanol 95:5); vmax (ATR)/cm"1: 2939, 1623, 1600, 1566, 1524, 1505, 1483, 1461 , 1396, 1342, 1325, 1263, 1230, 1 189, 1167, 1112, 1086, 1047, 994, 889, 873, 863, 828, 816, 762, 737, 698, 675; 1H- NMR (300 MHz, CDCI3): δ 3.44 (3 H, s, NCH3), 3.64 (3 H, s, 5-OCH3), 3.75 (3 H, s, 4-OCH3), 3.95 (3 H, s, 4-OCH3), 6.9-7.0 (2 H, m, 2-H, 6-H), 7.15 (1 H, d, 3 J 8.7 Hz, 5-H), 7.45 (1 H, dd, 3 J 8.7 Hz, 4 J 2.2 Hz, 6-H), 7.50 (1 H, s, 2-H), 7.78 (1 H, d, 3 J 2.2 Hz, 2-H); 1¾-NMR (75.5 MHz, CDCI3): δ 32.1 (NCH3), 55.7 (5-OCH3), 56.6 (4-OCH3), 60.5 (4-OCH3), 109.2 (C-6), 114.2 (C-5), 119.7 (C-2), 122.2 (C-1 ), 126.0 (C-3), 127.3 (C-2), 127.9 (C-5), 130.6 (C-1 ), 136.4 (C-6), 137.5 (C-4), 137.8 (C-2), 139.7 (C-3), 144.0 (C-4), 152.9 (C-4), 153.4 (C-5); m/z (%) 407 (42), 406 (46), 405 (87) [M+], 404 (77), 403 (100) [M+], 391 (23), 390 (71 ), 389 (62), 388 (87), 341 (58), 313 (58), 206 (33), 164 (26).
4) 1-Methyl-5-(3-amino-4-methoxyphenyl)-4-(3-ch!oro-4,5-dimethoxyph
bis(hydroch!oride) 5b x 2 HC!
Compound 5a (109 mg, 0.27 mmol) was dissolved in tetrahydrofuran (7.5 mL). Zn powder (107 mg, 1.36 mmol) was added followed by a mixture of cone. HCI (230 μί) in tetrahydrofuran (1 mL). After stirring for 15 mtn at room temperature the reaction mixture was poured into water and treated with aqueous NaHC03 to adopt pH 8. The water phase was extracted with ethyl acetate and the organic phase was dried over Na2S04, filtered and the filtrate was concentrated in vacuum. The residue thus obtained was purified by column chromatography (silica gel 60, 5% methanol/ethyl acetate, Rf = 0.66) giving crude 5b. This crude product was dissolved in dichloromethane (5 mL) and treated with 3M HCI/dioxane (1 mL). After stirring for 15 min the volatiles were removed and the oily residue was recrystallised from an ethanol/n-hexane mixture to leave the bis(hydrochloride) salt of 5b.
Yield: 42 mg (0.095 mmol, 40%); colorless solid of mp 80-183°C; UV (MeOH) max (ε) 255 (14940); vmax (ATR)/cm"1: 3009, 2781 , 2578, 1635, 1552, 1517, 1497, 1445, 1409, 1304, 1271 , 1 147, 1113, 1047, 1025, 998, 830, 762, 739, 721 ; 1H-NMR (300 MHz, DMSO-c/6): δ 3.63 (3 H, s, NMe), 3.69 (3 H, s, 5-OMe), 3.75 (3 H, s, 4-OMe), 3.92 (3 H, s, 4-OMe), 7.08 (1 H, d, 4J 2.1 Hz, 6-H), 7.16 (1 H, d, 4J 2.1 Hz, 2-H), 9.36 (1 H, s, 2-H); "C-NMR (75.5 MHz, DMSO-de): δ 33.9 (NMe), 56.1 (5-OMe), 56.2 (4-OMe), 60.4 (4-OMe), 110.7 (C-6), 1 12.4 (C-
5) , 1 17.6 (C-1 ), 1 19.6 (C-2), 123.6 (C-3), 127.3 (C-3), 127.6 (C-5), 129.7 (C-1 ), 135.7 (C-2), 145.0 (C-4), 153.5 (C-5); m/z (%) 375 (20) [M+], 374 (15), 373 (54) [M+], 358 (25), 296 (15),
252 (25), 237 (16), 70 (14), 61 (23), 43 (100).
Example 2: Synthesis of further compounds according to the invention
1 ) N-[(toluene-4-sulfonyl)-(3-bromo-4,5-dimethoxyphenyl)methyl]formamide 3b Analogously to the synthesis of 3a, compound 3b (4.78 g, 10.81 mmol, 56%) was obtained from 5-bromoveratraidehyde (5.67 g, 23.14 mmo!), para-toluenesulfinic acid (3 g, 19.29 mmol), camphorsu!fonic acid (110 mg, 0.47 mmol) and formamide (10 mL); colorless solid of mp 162-163 °C; vmax (ATR)/cm"1 3197, 3104, 2945, 1688, 1595, 1569, 1490, 1470, 1422, 1403, 1308, 1301 , 1290, 1279, 1250, 1229, 1207, 1 144, 1 121 , 1078, 1049, 998, 832, 769, 705, 688; H N R (300 MHz, CDC!3) £2.41 (3 H, s), 3.82 (6 H, s), 6.17 (1 H, s), 6.99 (1 H, d, J = 2.1 Hz), 7.17 (1 H, d, J = 2.1 Hz), 7.31 (1 H, d, J = 8.5 Hz), 7.67 (2 H, d, J = 8.5 Hz), 7.97 (1 H, s); 13C NMR (75.5 MHz, CDCI3) £ 21.9, 56.4, 61.0, 70.7, 113.3, 118.0, 125.9, 127.3, 129,9, 130.2, 133.1 , 146.2, 147.9, 154.0, 161.3; m/z (El) 274 (28), 273 (45), 272 (37), 271 (40), 242 (27), 192 (100), 156 (30), 92 (56), 91 (55), 65 (36).
2) N-[(Toiuene-4-sulfonyi)-(3,4-dimethoxy-5 nitrophenyl)methyl]formamide 3c Analogously to the synthesis of 3a, compound 3c (2.63 g, 6.68 mmol, 35%) was obtained from 5-nitroveratraldehyde (4.85 g, 22.99 mmol), para-toluenesulfinic acid (2.96 g, 19.03 mmol), camphorsulfonic acid (1 10 mg, 0.47 mmol) and formamide (10 mL); colorless solid of mp 133 °C; vmax (ATR)/cm'1 3194, 2888, 1662, 1539, 1518, 1389, 1354, 1319, 1302, 1291 , 1212, 1271 , 1084, 1074, 1053, 991 , 922, 858, 819; H NMR (300 MHz, CDCI3) £2.41 (3 H, s), 3.87 (3 H, s), 3.95 (3 H, s), 6.33 (1 H, d, J = 10.6 Hz), 7.26 (1 H, d, J = 2.1 Hz), 7.31 (1 H, d, J = 8.5 Hz), 7.43 (1 H, d, J = 2.1 Hz), 7.70 (2 H, d, J = 8.5 Hz), 8.06 (1 H, s), 8.9-9.0 (1 H, m); 3C NMR (75.5 MHz, CDCI3) £21.7, 56.5, 62.0, 69.9, 1 16.4, 1 17.2, 125.8, 129.4, 130.1 , 132.3, 143.9, 144.5, 146.1 , 154.1 , 160.3; m/z (El) 196 (7), 155 (9), 91 (100), 65 (58).
3) 3-Bromo-4,5-dimethoxypheny!(tosyl)methyl isocyanide 4b
Compound 3b (4.75 g, 10.75 mmol) was suspended in dry DME (100 mL) and cooled to -10 °C. POCI3 (3.1 mL, 33.1 moi) was added and a mixture of Et3N (7.5 mL, 53.8 mmol) in DME (10 mL) was dropped s!owly to the reaction mixture. After stirring for 2 h at -5 °C, the reaction mixture was poured into ice water. The water phase was extracted with ethyl acetate, the organic phase was washed with saturated aqueous NaHC03 and brine, dried over Na2S04, filtered and concentrated in vacuum. By refrigeration over night a brown solid crystallized from the residue, which was collected and dried in vacuum. Yield: 2.75 g (6.71 mmoi, 62%); brown solid of mp 109-110 °C; vmax (ATR)/cnrf 1 2915, 2135, 1593, 1569, 1489, 1452, 1420, 1325, 1295, 1275, 1238, 1199, 1 136, 1081 , 1048, 1003, 862, 826, 759, 705, 670; 1H NMR (300 MHz, CDCI3) 2.47 (3 H, s), 3.81 (3 H, s), 3.86 (3 H, s), 5.46 (1 H, s), 6.81 (1 H, d, J = 2.2 Hz), 7.01 (1 H, d, J = 2.2. Hz), 7.35 (2 H, d, J - 8.3 Hz), 7.64 (2 Hr d, J = 8.3 Hz); 3C NMR (75.5 MHz, CDCI3) £ 21.8, 56.2, 60.7, 75.5, 1 1 1 .5, 117.8, 123.2, 125.0, 129.9, 130.0, 130.5, 146.9, 148.5, 153.6, 166.6; m/z (El) 256 (52), 254 (45), 244 (78), 242 (100), 200 (30), 123 (48), 91 (31 ).
4) 3,4,-Dimeihoxy-5-niirophenyi(tosyl)methyl isocyanide 4c
Compound 3c (2.63 g, 6.68 rnmoi) was suspended in dry DME (100 mL) and cooled to -10 °C. POCI3 (3.78 mL, 40.4 mol) was added and a mixture of Et3N (7.5 mL, 66.6 mmol) in DME (10 mL) was dropped slowly to the reaction mixture. After stirring for 2 h at -5 "C, the reaction mixture was poured into ice water. The water phase was extracted with ethyl acetate, the organic phase was washed with saturated aqueous NaHC03 and brine, dried over Na2S04, filtered and concentrated in vacuum. By refrigeration over night a yellow solid crystallized from the residue, which was collected and dried in vacuum. Yield: 520 mg (1.51 mmol, 23%); yellow solid of mp 134 °C (dec); vmax (ATR)/cnf1 2949, 2140, 1593, 1538, 1494, 1453, 1360, 1337, 1315, 1285, 1248, 1 184, 1 156, 1145, 1082, 1065, 988, 921 , 819, 784, 701 , 667; 1H NMR (300 MHz, CDCI3) £2.48 (3 H, s), 3.91 (3 H, s), 4.00 (3 H, s), 5.53 (1 H, s), 7.12 (1 H, d, J = 2.2 Hz), 7.18 (1 H, d, J = 2.2 Hz), 7.38 (2 H, d, J = 8.4 Hz), 7.67 (2 H, d, J - 8.4 Hz); 13C NMR (75.5 MHz, CDCI3) £21.9, 56.7, 62.3, 75.3, 115.3, 1 16.3, 122.3, 129.7, 129.8, 130.4, 144.6, 144.8, 147.4, 154.4, 167.5; m/z (El) 375 (6) [M+ - 1], 344 (12), 278 (23), 262 (22), 221 (100), 21 1 (47), 155 (32), 139 (38).
5) 1-Methyl-4-(3,-chloro-4',5!-dimethoxyphenyl)-5-(3"-fluoro-4,'-methoxyphenyl)-imidazole 5c
Analogously to the synthesis of compound 5a, compound 5c was prepared from 3-fluoro-4- methoxybenzaldehyde (65 mg, 0.42 mmol), 33% MeNH2/ethanol (260 uL, 2.10 mmol) and acetic acid (150 μί) in boiling ethanol (15 mL) giving the imine intermediate, which was treated with compound 4a (153 mg, 0.42 mmol), dissolved in DME (10 mL), and K2C03 (500 mg, 3.62 mmol). After workup, the residue was purified by column chromatography (silica gel 60). Yield: 150 mg (0.38 mmol, 91 %); colorless gum; Rf = 0.27 (ethyl acetate/m ethanol 95:5);vmax (ATR)/cm*1 2937, 2837, 1601 , 1553, 1512, 1487, 1462, 1419, 1301 , 1067, 1233, 1166, 1131 , 1046, 1022, 999; 896, 873, 829, 815, 761 , 734, 719, 656; 1H NMR (300 MHz, CDCI3) £3.39 (3 H, s), 3.61 (3 H, s), 3.75 (3 H, s), 3.87 (3 H, s), 6.9-7.0 (5 H, m), 7.45 (1 H, s); 13C NMR (75.5 MHz, CDCI3) £ 31.9, 55.6, 56.1 , 60.5, 109.0, 1 13.7, 118.0, 118.2, 119.4, 122.4, 122.5, 126.9, 127.5, 127.8, 131.0, 136.7, 137.2, 143.6, 148.0, 148.1 , 150.5, 153.2, 153.8; m/z (El) 379 (15), 378 (65) [M+], 377 (41 ), 376 (100) [M+], 363 (26), 361 (67). 6) 1-Methy[-4-(3'-chioro-4 5,-dimethoxyphenyl)-5-(3'!-fluoro-4,'-meihoxyphenyl)-im hydrochloride 5c x HCI Compound 5c {150 mg, 0.38 mmol) was dissolved in DCM (5 mL) and treated with 3M HCI/dioxane (1 mL). After stirring for 15 mm the solvent was removed and the oily residue was recrystallised from an DC /n-hexane mixture giving the hydrochloride sait of 5c. Yield: 106 mg (0.25 mmol, 66%); colorless solid of mp 185-187 °C;UV {MeOH) (ε) 264 (10380); vmax (ATR)/cm'1 3387, 2941 , 2840, 2605, 1623, 1553, 1523, 1498, 1464, 1456, 1422, 1303, 1272, 1232, 1 134, 1 1 17, 1048, 1019, 997, 872, 844, 817, 760; 1H NMR (300 MHz, DMSO-d6) 3.63 (3 H, s), 3.70 (3 H, s), 3.75 (3 H, s), 3.92 (3 H, s), 7.02 (1 H, d, J = 2.1 Hz), 7.17 (1 H, d, J = 2.1 Hz), 7.2-7.3 (1 H, m), 7.39 (1 H, t, J = 17.2 Hz), 7.51 (1 H, dd, J = 12.0 Hz, J = 2.0 Hz), 9.29 (1 H, s); 13C NMR (75.5 MHz, DMSO-d6): δ 33.8, 56.0, 56.2, 60.3, 1 10.8, 114.6, 1 17.9, 118.0, 1 18.3, 1 18.5, 1 19.6, 124.0, 127.2, 128.1 , 128.5, 128.8, 135.8, 144.9, 148.7, 148.9, 149.8, 153.0, 153.5.
7) 1-Methyl-4-(3'-chioro-4',5'-dimethoxyphenyl)-5-(4"-N,N-dimethylaminophenyl)-imidazole 5d Analogously to the synthesis of compound 5a, compound 5d was prepared from 4-N,N- dtmethylaminobenzaldehyde (63 mg, 0.42 mmol), 33% MeNH2/ethanol (260 pL, 2.10 mmol) and acetic acid (150 pL) in boiling ethanol (15 mL) giving the imine intermediate, which was treated with compound 4a (153 mg, 0.42 mmol), dissolved in DME (10 mL), and K2C03 (500 mg, 3.62 mmol). After workup, the residue was purified by coSumn chromatography (silica gel 60). Yield: 140 mg (0.38 mmol, 91 %); colorless oil; Rf = 0.67 (ethyl acetate/methanol 95:5); max (ATR)/cm"1 2935, 2825, 1612, 1552, 1516, 1485, 1397, 1357, 1316, 1261 , 1228, 1 187, 1 165, 1108, 1047, 1000, 944, 881 , 857, 818, 762, 721 , 660; 1H NMR (300 MHz, CDCI3) δ 2.96 (6 H, s), 3.39 (3 H, s), 3.59 (3 H, s), 3.76 (3 H, s), 6.72 (2 H, d, J = 8.9 Hz), 7.00 (1 H, d, J = 1.9 Hz), 7.11 (2 H, d, J = 8.9 Hz), 7.16 (1 H, d, J = 1.9 Hz), 7.46 (1 H, s); 13C NMR (75.5 MHz, CDCI3) £31.8, 40.2, 55.5, 60.5, 108.8, 1 12.3, 1 16.8, 119.2, 127.7, 129.7, 131.3, 131.6, 135.9, 136.7, 143.2, 150.5, 153.0; m/z (El) 373 (36) [M*], 371 (100) [M+], 356 (42), 281 (10), 72 (19), 59 (34).
8) 1-Methyi-4-(3'-chloro-4',5'-dimethoxyphenyl)-5-(4'!-N,N-dimethylaminophenyl)-imidazole bis(hydroch!oride) 5d x 2 HCi Compound 5d (140 mg, 0.38 mmol) was dissolved in DC (5 mL) and treated with 3M HCl/dioxane (1 mL). After stirring for 10 min the solvent was evaporated and the residue crystallized from ethanol/n-hexane. Yield: 77 mg (0.17 mmol, 46%); colorless solid of mp 189-193 °C (dec); UV (MeOH) Xma>l (ε) 265 (20800); vmax (ATR)/cm"1 3356, 3022, 2956, 2835, 2452, 1620, 1595, 1551 , 1499, 1470, 1422, 1319, 1279, 1231 , 1 190, 1160, 1129, 1052, 1020, 989, 897, 859, 842, 756, 696; 1H N R (300 MHz, DMSO-d6) 3.00 (6 H, s), 3.62 (3 H, s), 3.69 (3 H, s), 3.74 (3 H, s), 6.98 (2 H, d, J = 8.8 Hz), 7.07 (1 H, d, J = 2.1 Hz), 7.21 (1 H, d, J = 2.1 Hz), 7.33 (2 H, d, J = 8.8 Hz), 9.37 (1 H, s); 3C NMR (75.5 MHz, DMSO-d6) £33.9, 40.2, 56.1 , 60.4, 107.8, 113.2, 1 19.5, 123.8, 127.0, 127.2, 130.8, 131.6, 135.4, 144.9, 150.8, 153.5.
9) 1-Methyl-4-(3,-chloro-4',5'-dimethoxyphenyl)-5-(4"-ethoxy-3"-nitrophenyl)-imidazole 5e
A mixture of 4-ethoxy-3-nitrobenza!dehyde (82 mg, 0.42 mmol) and 33% MeNH2/ethanoi (260 pL, 2.10 mmol) in ethanol (15 mL) was treated with AcOH (150 μΐ_, 2.63 mmol) and refluxed for 2 h. After cooling down to room temperature, 4a (153 mg, 0.42 mmol) and K2C03 (500 mg, 3.62 mmol) were added and the reaction mixture was refluxed for 5 h. The solvent was evaporated, the residue diluted with ethyl acetate, washed with water and brine, dried over Na2S04, filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60, ethyl acetate/methanol 9:1 ) giving the product as an orange oil. Yield: 170 mg (0.34 mmoi, 81 %); vmax (ATR)/crn 1: 2938, 1622, 1600, 1553, 1527, 1506, 1484, 1353, 1286, 1263, 1 110, 1045, 998, 870, 829, 817, 762, 739, 656, 635; 1H NMR (300 MHz, CDCI3): δ 1.43 (3 H, t, 3 J 7.0 Hz, Me), 3.44 (3 H, s, NMe), 3.63 (3 H, s, 4'-OMe), 3.75 (3 H, s, 5'-OMe), 4.18 (2 H, q, 3 J 7.0 Hz, OCH2), 6.9-7.0 (2 H, m, 2'-H, 6!-H), 7.12 (1 H, d, 3 J 8.7 Hz, 5"-H), 7.41 (1 H, dd, 3 J 8.7 Hz, 4 J 2,2 Hz, 6"-H), 7.49 (1 H, s, 2-H), 7.75 (1 H, d, J 2.2 Hz, 2"-H); 13C NMR (75.5 MHz, CDCI3): δ 14.3 (Me), 32.1 (NMe), 55.7 (5'-OMe), 60.5 (41- OMe), 65.5 (OCH2), 109.1 (C-61), 1 15.0 (C-5"), 1 19.6 (C-2'), 121.9 (C-1 "), 126.1 (C-5), 127.2 (C-6"), 127.9 (C-3'), 130.6 (C-1 '), 136.2 (C-2"), 137.4 (C-4), 137.8 (C-2), 139.9 (C-3"), 143.9 (C-4'), 152.2 (C-5'), 153.3 (C-4"); m/z (%) 419 (36) [M+], 417 (100) [M+], 402 (53).
10) 1 -Methyl-5-(3"-amino-4"-ethoxyphenyl)-4-(3'-chloro-4',5'-dimethoxyphenyl)-imidazole 5f x 2HCI
Compound 5e (140 mg, 0.34 mmol) was dissolved in THF (7,5 mL). Zn powder (110 mg, 1.68 mmol) was added followed by a mixture of cone. HCI (243 μί) in THF (1 mL). After stirring for 15 min at room temperature the reaction mixture was poured onto water and basified with aqueous NaHC03 to ca. pH 8. The water phase was extracted with ethyl acetate and the organic phase was dried over Na2S04, filtered and the filtrate was concentrated in vacuum. The residue was purified by column chromatography (silica gel 60, 10% methanoi/ethyl acetate, Rf = 0.63) giving the aniline intermediate. This compound was dissolved in DCM (5 mL) and treated with 3M HCI/dioxane (1 mL). After stirring for 15 min the solvent was removed and the residue was recrystailized from a DCM/n-hexane mixture. Yield: 97 mg (0.21 mmol, 62%); colorless solid of m.p. >150°C (dec); vmax (ATR)/cm"1: 3392, 2976, 2833, 2538, 1631 , 1550, 1514, 1495, 1466, 1396, 1302, 1268, 1237, 1 142, 1 1 15, 1044, 996, 850, 816, 762, 722; 1H NMR (300 MHz, DMSO-d6): δ 1.40 (3 H, t, 3J 6.9 Hz, Me), 3.63 (3 H, s, NMe), 3.70 (3 H, s, 4'-OMe), 3.75 (3 H, s, 5 -O e), 4.21 (2 H, q, 3 J 6.9 Hz, OCH2), 7.07 (1 H, d, 4 J 2.1 Hz, 6'-H), 7.19 (1 H, d, 4 J 2.1 Hz, 2'-H), 7.3-7.4 (2 H, m, 5"-H, 6"H), 7.45 (1 H, s, 2"-H), 9.41 (1 H, s, 2-H); 13C NMR (75.5 MHz, DMSO-d6): δ 14.4 (Me), 34.0 (NMe), 56.2 (5'-O e), 60.4 (4'-OMe), 64.6 (OCH2), 110.8 (C-6'), 1 13.7 (C-5"), 1 17.5 (C- 1 "), 1 19.6 (C-2'), 123.5 (C-5), 127.3 (C-3'), 127.7 (C-f), 129.3 (C-4), 135.8 (C-2), 145.0 (C- 4'), 151.7 (C-5'), 153.5 (C-4"); m/z (%) 388 (35) [M+], 386 (100) [M+], 371 (36), 36 (98).
1 1 ) 1-Methyl-4-(3,-chloro-4',5,-dimethoxyphenyl)-5-(3"-fluoro-4"-ethoxyphenyi)-imidazole 5g x HCI
A mixture of 3-fluoro-4-ethoxybenzaldehyde (124 mg, 0.74 mmol) and 33% MeNH2/ethanol (460 pL, 3.76 mmol) in ethanol (15 mL) was treated with AcOH (260 pL, 4.63 mmo!) and ref!uxed for 2 h. After cooling down to room temperature, 4a (270 mg, 0.74 mmol) and K2C03 (500 mg, 3.62 mmol) were added and the reaction mixture was ref!uxed for 5 h. The solvent was evaporated, the residue diluted with ethyl acetate, washed with water, dried over Na2S04, filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60, ethy! acetate/methanoi 95:5) giving the imidazole as a colorless oil. This oil was dissolved in DCM (5 mL) and treated with 3 M HCI in dioxane (1 mL). After stirring for 5 min the solvent was evaporated and the residue crystallised from DCM/n-hexane. Yield: 210 mg (0.49 mmol, 66%), colorless solid;vmax (ATR)/cm"1: 3391 , 3166, 2946, 2841 , 2727, 1627, 1554, 1523, 1499, 1477, 1423, 1395, 1302, 1269, 1230, 1 193, 1136, 1 1 17, 1050, 998, 888, 844, 814, 778, 753; H NMR (300 MHz, acetone-c/6): δ 1.44 (3 H, t, 3 J 7.0 Hz), 3.75 (3 H, s), 3.77 (3 H, s), 3.88 (3 H, s), 4.25 (2 H, q, 3 J 7.0 Hz), 7.01 (1 H, d, 4J 2.1 Hz), 7.35 (1 H, t, 3JHF 18.0 Hz), 7.56 (1 H, dd, 3J 11.7 Hz, 4J 1.9 Hz), 9.71 (1 H, s); 13C NMR (75.5 MHz, acetone-cf6): δ 15.0, 34.9, 57.1 , 60.8, 65.7, 11 1.8, 113.8, 116.3, 1 19.2, 1 19.3, 119.6, 119.9, 120.6, 122.4, 124.4, 124.5, 128.6, 129.2, 130.1 , 134.6, 136.6, 146.4, 149.8, 149.9, 151.7, 154.8, 155.0; m/z {%) 392 (37) [M+], 390 (100) [M+], 377 (21 ), 375 (65). 12) 1 -Methyl-4-(3'-chloro-4',5'-dimethoxyphenyl)-5-(N-m
5i Analogously to the synthesis of 5a, compound 5i was obtained from the imine of W-rnethyl-3- chloroindol-5-carboxaldehyde (81 mg, 0.42 mmoi), 33% MeNH2/ethanol (260 μί_, 2.10 mmol) and acetic acid (150 μί_) in boiling ethanoi (15 ml_), which was treated with 4a (153 mg, 0.42 mmol) and K2C03 (500 mg, 3.62 mmoi). Yie!d: 160 mg (0.39 mmol, 93%); colorless oil; Rf = 0.62 (ethyl acietate/methanol 95:5);vmax (ATR)/cm~1: 2937, 2830, 1600, 1565, 1504, 1476, 1463, 1397, 1314, 1261 , 1240, 1 166, 1109, 1047, 999, 972, 907, 883, 856, 831 , 803, 727, 700, 686, 656; H-NMR (300 MHz, CDCI3): δ 3.41 (3 H, s, 1-NMe), 3.52 (3 H, s, 5'-OMe), 3.75 (6 H, s, 1 "-NMe, 4'-OMe), 7.02 (1 H, d, J 2.0 Hz, 6'-H), 7.06 (1 H, s, 2"-H), 7.09 (1 H, d, 4J 2.0 Hz, 2!-H), 7.14 (1 H, dd, 3J 8.5 Hz, 4J 1.5 Hz, 6"-H), 7.35 (1 H, d, 3J 8.5 Hz, 7"-H), 7.52 (1 H, s, 2-H), 7.57 (1 H, d, 4J 1.5 Hz, 4"-H); 13C-NMR (75.5 MHz, CDCI3): δ 32.0 (1-NMe), 33.0 (1 "-NMe), 55.6 (5"-OMe), 60.5 (4!-OMe), 104.6 (C-3"), 109.0 (C-61), 1 10.3 (C-7"), 119.4 (C-2'), 120.5 (C-4"), 121.5 (C-5"), 125.1 (C-6"), 126.0, 126.3 (C-2"), 127.7 (C-3'), 129.8 (C- 5), 131.5 (C-r), 135.7 (C-7a"), 136.3 (C-4), 136.9 (C-2), 143.5 (C-4'), 153.1 (C-5'); m/z (El) 417 (72) [Ml 415 (100) [M+], 402 (43), 400 (55). 13) 1 -Methyl-4-(3'-chioro-4!,5'-dimethoxyphenyi)-5-(N-methyi-3"-chloroindol-5"-yl)-imidazole- hydrochloride 5i x HCI
Compound 5i (160 mg, 0.39 mmol) was dissolved in DCM (5 mL) and treated with 3M HCI/dioxane (1 mL). After stirring at room temperature for 10 min the solvent was evaporated in vacuum and the residue was recrystallised from DCM/n-hexane. Yield: 98 mg (0.20 mmol, 51 %); colorless solid; mp. 198°C; UV (MeOH) Xmax (ε) 230 (30800);vm3x (ATR)/crrf1: 3392, 2941 , 2845, 2602, 1622, 1565, 1542, 1493, 1464, 1456, 1318, 1239, 1115, 1046, 996, 971 , 871 , 852, 803; 1H-NMR (300 MHz, DMSO-de): δ 3.61 (6 H, s, 1-NMe, 5'-OMe), 3.72 (3 H, s, 4 -OMe), 3.86 (3 H, s, 1 "-NMe), 6.99 (1 H, d, 4J 2.1 Hz, 6'-H), 7.20 (1 H, d, 4J 2.1 Hz, 2'-H), 7.34 (1 H, dd, 3 J 9.4 Hz, 4 J 1.6 Hz, 6,!-H), 7.7-7.8 (3 H, m, 2"-H, 4"-H, 7"-H), 9.32 (1 H, s, 2- H); 13C-NMR (75.5 MHz, DMSO-d6): 5 33.0 (1-NMe), 33.9 (Γ-NMe), 56.0 (5"-OMe), 60.3 (4 - OMe), 102.7 (C-3"), 1 10.8 (C-6'), 1 1 1.8 (C-7"), 1 17.0 (C-5"), 1 19.5 (C-2'), 120.4 (C-4"), 124.1 (C-3a,!), 124.5 (C-6"), 125.1 (C-7a"), 127.2 (C-3'), 127.9 (C-5), 128.3 (C-2"), 130.9 (C- 1 '), 135.6 (C-2), 136.0 (C-4), 144.8 (C-4'), 153.4 (C-5'); m/z (El) 417 (100) [M+ - 2HCI], 415 (68) [M+ - 2HCI], 402 (66), 400 (96).
14) 1 -Methy!-4-(3'-bromo-4',5'-dimethoxyphenyl)-5-(4"-methoxy-3"-nitrophenyl)-imidazole 6a A mixture of 4-methoxy-3-nitrobenzaldehyde (76 mg, 0.42 mrnoi) and 33% MeNH2/ethanoi (260 μΙ_, 2.10 mmol) in ethano! (15 mL) was treated with AcOH (150 iL) and refluxed for 2 h. After cooling down to room temperature, compound 4b (172 mg, 0.42 mmol) dissolved in DME (10 mL) and K2C03 (500 mg, 3.62 mmol) was added and the reaction mixture was refluxed for 3 h. The solvent was evaporated, the residue diluted with ethyl acetate, washed with water and brine, dried over Na2S04, filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60; elution with ethyi acetate to 5% methanol/ethyl acetate). Yield: 100 mg (0.22 mmo!, 52%); yellow oil; f = 0.36 (ethyl acetate); vmax (ATR)/cm"1 2937, 2832, 1622, 1599, 1548, 1526, 1506, 1480, 1462, 1351 , 1263, 1231 , 1 185, 1 165, 1110, 1092, 1042, 998, 909, 889, 868, 824, 808, 760, 728; 1H NMR (300 MHz, CDCIs) 3.46 (3 H, s), 3.64 (3 H, s)), 3.75 (3 H, s), 3.97 (3 H, s), 6.97 (1 H, s), 7.12 (1 H, s), 7.16 (1 H, d, J = 8.7 Hz), 7.46 (1 H, d, J = 8.7 Hz), 7.51 (1 H, s), 7.80 (1 H, s); 13C NMR (75.5 MHz, CDCI3) £ 32.2, 55.7, 56.7, 60.4, 1 10.0, 114.2, 117.4, 122.2, 122.5, 126.1 , 127.4, 131.2, 136.5, 137.5, 137.9, 139.7, 145.0, 152.9, 153.3; m/z (El) 448 (100) [M+], 446 (97) [M+], 433 (37), 431 (41 ), 206 (27), 164 (23).
15) 1 ~Methyl-5-(3"~am!no-4"-methoxyphenyl)-4-(3>-bromo-4I,5'-dimethoxyphenyl)-imidazole 6b
Analogously to 5b, compound 6a (100 mg, 0.22 mmol) was reduced by Zn powder (72 mg, 1.1 1 mmol) and cone. HCI (160 μί.) in THF (8.5 mL). After workup the residue was purified by column chromatography (silica gel 60; ethyl acetate/methanol 95:5). Yield: 83 mg (0.20 mmol, 91 %); colorless solid of mp 166-169 °C; R, = 0.48 (ethyl acetate); UV (MeOH) max (ε) 274 (12760); vmax (ATR)/cm"1 3458, 3365, 3192, 2935, 2833, 1616, 1597, 1546, 1510, 1483, 1462, 1420, 1372, 1318, 1279, 1245, 1223, 1174, 1 109, 1040, 1024, 997, 868, 804, 759, 658; 1H NMR (300 MHz, DMSO-d6) £3.41 (3 H, s), 3.57 (3 H, s), 3.67 (3 H, s), 3.82 (3 H, s), 4.91 (2 H, s), 6.55 (1 H, d, J = 8.2 Hz), 6.61 (1 H, s), 6.94 (1 H, d, J = 8.2 Hz), 7.09 (1 H, s), 7.26 (1 H, s), 7.71 (1 H, s); 13C NMR (75.5 MHz, DMSO-dg) £31.6, 55.4, 60.0, 109.4, 1 10.9, 1 15.3, 1 16.4, 118.2, 120.8, 122.2, 129.8, 132.7, 134.2, 138.3, 143.5, 146.7, 152.8; m/z (El) 418 (100) [M+3, 416 (96) ijvT], 403 (78), 401 (85), 307 (17), 169 (22).
16) 1 -MethyI-5-(3"-amino-4"-methoxyphenyi)-4-(3'-bromo-4',5'-dimethoxyphenyl)-imidazole bis(hydrochloride) 6b x 2HCI
Compound 6b (61 mg, 0.15 mmol) was dissolved in DCM and treated with 3M HC!/dioxane (1 mL). After stirring for 15 min at room temperature the solvent was removed and the residue recrystaliized from an ethanol/n-hexane mixture. Yield: 72 mg (0.15 mmol, 100%); colorless solid of mp 198-200 °C; vmax (ATR)/crrf1 3011 , 2801 , 2567, 1633, 1549, 1515, 1494, 1446, 1405, 1305, 1272, 1145, 1 113, 1041 , 1022, 994, 867, 852, 829, 718; 1H NMR (300 MHz, DMSO-de): <? 3.63 (3 H, s), 3.69 (3 H, s), 3.73 (3 H, s), 3.93 (3 H, s), 7.18 (1 H, d, J = 2.0 Hz), 7.22 (1 H, d, J = 2.0 Hz), 7.3-7.4 (3 H, m), 9.37 (1 H, s); 13C NMR (75.5 MHz, DMSO-d6) 333,9, 56.1 , 60.2, 1 11.3, 112.5, 117.0, 117.6, 122.4, 124.2, 127.5, 129.6, 135.8, 146.0, 151.6, 153.3.
17) 1-Meihy 4-(3,-bromo-4\5'-dimethoxyphenyi)-5-(3,'-fluoro-4"-methoxyphenyl)-imidazole 6c
Analogously to the synthesis of 5a, compound 6c was prepared from 3-fluoro-4- methoxybenzaldehyde (65 mg, 0.42 mmol), 33% MeNH2/ethanoi (260 pL, 2.10 mmol) and acetic acid (150 pL) in boiling ethanol (15 mL) giving the imine intermediate, which was treated with compound 4b (172 mg, 0.42 mmol), dissolved in DME (10 mL), and K2C03 (500 mg, 3.62 mmol). After workup, the residue was purified by column chromatography (silica gel 60; elution with ethyl acetate to 5% methanol/ethyl acetate). Yield: 135 mg (0.32 mmol, 76%); colorless gum; Rf ~ 0.67 (ethyl acetate); UV (MeOH) max (ε) 275 (12280); vmax (ATR cm"1 2934, 2832, 1598, 1548, 151 1 , 1483, 1462, 1418, 1300, 1265, 1233, 1214, 1 166, 1131 , 1109, 1041 , 1022, 997, 895, 865, 816, 807, 760, 656; 1H NMR (300 MHz, CDCi3) δ 3.45 (3 H, s), 3.58 (3 H, m), 3.68 (3 H, s), 3.90 (3 H, s), 7.01 (1 H, s), 7.16 (1 H, s), 7.2-7.4 (3 H, m), 7.78 (1 H, s); 13C NMR (75.5 MHz, CDCI3) 31.8, 55.4, 56.1 , 60.1 , 109.6, 1 14.3, 1 16.5, 1 17.9, 1 18.2, 121.1 , 122.3, 122.4, 127.5, 127.6, 127.7, 132.3, 135.2, 138.1 , 143.8, 147,5, 147.7, 149.8, 153.0, 153.1 ; m/z (El) 421 (51 ) [M+], 419 (49) [M+], 406 (23), 404 (22), 233 (11 ), 175 (31 ), 1 17 (75), 59 (100).
18) 1-Methyl-4-(3'-bromo-4',5,-dimethoxyphenyl)-5-(3"-fluoro-4"-methoxyphenyl)-imidazole hydrochloride 6c x HCi Compound 6c (135 mg, 0.32 mmol) was dissolved in DCM (5 mL) and treated with 3M HCI/dioxane (1 mL). After stirring for 15 min the solvent was removed and the oily residue was recrystallised from an DCM/n-hexane mixture giving the hydrochloride salt. Yield: 91 mg (0.20 mmol, 63%); colorless solid of mp 103-106 °C (dec); UV (MeOH) max (ε) 270 (1 1680); vmax (ATR)/cm"1 3413, 3012, 2936, 2841 , 2626, 1625, 1547, 1523, 1493, 1463, 1421 , 1304, 1271 , 1234, 1203, 1 134, 1116, 1041 , 994, 869, 849, 818, 760; 1H NMR (300 MHz, DMSO-d6) £3.64 (3 H, s), 3.68 (3 H, s), 3.73 (3 H, s), 3.92 (3 H, s), 7.16 (1 H, d, J = 2.0 Hz), 7.19 (1 H, d, J = 2.0 Hz), 7.3-7.4 (1 H, m), 7.39 {1 H, t, J = 17.2 Hz), 7.51 (1 H, dd, J = 12.0 Hz, J = 2.0 Hz), 9.28 (1 H, s); 3C NMR (75.5 MHz, CDCI3) £34.3, 56.5, 56.7, 60.7, 111.9, 1 15.1 , 117.4, 118.3, . 118.4, 118.8, 119.0, 123.0, 125.1 , 128.6, 128.8, 129.3, 136.3, 146.4, 149.2, 149.3, 150.2, 153.5, 153.8.
19) 1-Methyl-4-(3'-bromo-4\5'-dimethox
6d
Analogously to the synthesis of 5a, compound 6d was prepared from 4-N,N- dimethyiaminobenzaldehyde (63 mg, 0.42 mmol), 33% eNH2/ethanol (260 pL, 2.10 mmoi) and acetic acid (150 μΙ_) in boiling ethanoi (15 mL) giving the tmine intermediate, which was treated with compound 4b (172 mg, 0.42 mmol), dissolved in DME (10 mL), and K2C03 (500 mg, 3.62 mmol). After workup, the residue was purified by column chromatography (silica gel 60). Yield: 140 mg (0.34 mmol, 81 %); colorless gum; Rf = 0.65 (ethyl acetate/methanol 95:5); vmax (ATR)/cm"1 2933, 2828, 1612, 1546, 1515, 1481 , 1462, 1357, 1314, 1258, 1228, 187, 1164, 1108, 1039, 999, 944, 873, 859, 821 , 806, 758, 738, 717, 659; 1H NMR (300 MHz, CDCI3) 2.94 (6 H, s), 3.39 (3 H, s), 3.56 (3 H, s), 3.74 (3 H, s), 6.72 (2 H, d, J = 8.9 Hz), 7.00 (1 H, d, J = 1.9 Hz), 7.10 (2 H, d, J - 8.9 Hz), 7.35 (1 H, d, J = 1.9 Hz), 7.46 (1 H, s); 13C NMR (75.5 MHz, CDCI3) £ 31.8, 40.2, 55.5, 60.3, 109.5, 1 12.3, 116.8, 117.2, 122.1 , 129.7, 131.3, 132.3, 135.8, 136.7, 144.2, 150.5, 152.9; m/z (El) 417 (54), 415 (68), 402 (28), 400 (26), 278 (54), 250 (58), 234 (63), 206 (83), 145 (84), 125 (87), 42 (100).
20) 1 -Methyl-4-(3'-bromo-4',5'-dimethoxyphenyl)-5-(4"-N,N-dimethylam!nopheny!)-imidazole bis(hydroch!oride) 6d x 2HCI
Compound 6d (140 mg, 0.34 mmol) was dissolved in DCM and treated with 3M HCI/dioxane (1 mL). After stirring for 15 min at room temperature the solvent was removed and the residue recrystallized from an ethano!/n-hexane mixture. Yield: 68 mg (0.14 mmol, 41 %); colorless solid of mp 189-193 °C (dec); UV (MeOH) max (ε) 265 (21220);vmax (ATR)/cm"1 3357, 2541 , 2451 , 1593, 1546, 1497, 1470, 1421 , 1316, 1277, 1230, 1190, 1159, 1129, 1046, 987, 843; 1H NMR (300 MHz, DMSO-d6) £ 2.99 (6 H, s), 3.63 (3 H, s), 3.68 (3 H, s), 3.73 (3 H, s), 6.94 (2 H, d, J = 8.9 Hz), 7.21 (2 H, s), 7.32 (2 H, d, J = 8.9 Hz), 9.35 (1 H, s); 13C NMR (75.5 MHz, DMSO-d6) £33.9, 56.1 , 60.2, 11 1.3, 112.9, 1 16.9, 122.3, 124.4, 126.9, 130.8, 131.5, 135.3, 145.9, 150.9, 153.3.
21 ) 1-Methyl-4-(3'-bromo-4',5'-dimethoxyphenyl)-5-(4"-ethoxy-3"-nitrophenyl)-imidazole 6e A mixture of 4-ethoxy-3-nitrobenzaidehyde (82 mg, 0.42 mmol) and 33% MeNH2/ethanol (260 μΙ_, 2.10 mmol) in ethanol (15 mL) was treated with AcOH (150 pL, 2.63 mmol) and refiuxed for 2 h. After cooling down to room temperature, 4b (172 mg, 0.42 mmol) and K2C03 (500 mg, 3.62 mmol) were added and the reaction mixture was refluxed for 5 h. The solvent was evaporated, the residue diluted with ethyl acetate, washed with water and brine, dried over Na2S04, filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60, ethyl acetate/methanol 9:1 ) giving the product as an orange oil. Yield: 170 mg (0.37 mmol, 88%); vmax (ATR)/cm"1: 2983, 2936, 1622, 1599, 1547, 1527, 1505, 1472, 1353, 1247, 1 1 10, 1039, 997, 865, 808, 759, 739, 655, 634; 1H NMR (300 MHz, CDCI3): δ 1.41 (3 H, t, 3J 7.0 Hz, Me), 3.42 (3 H, s, NMe), 3.60 (3 H, s, 4'~OMe), 3.72 (3 H, s, 5'-OMe), 4.16 (2 H, q, 3 J 7.0 Hz, OCH2), 6.92 (1 H, d, 4 J 2.0 Hz, 6'-H), 7.0-7.1 (2 H, m, 2'-H, 5"-H), 7.40 (1 H, dd, 3 J 8.7 Hz, 4 J 2.3 Hz, 6"-H), 7.47 (1 H, s, 2-H), 7.73 (1 H, d, 4 J 2.3 Hz, 21!-H); 13C NMR (75.5 MHz, CDCI3): δ 14.2 (Me), 32.1 (NMe), 55.6 (5 -OMe), 60.3 (4'-OMe), 65.5 (OCH2), 109.8 (C-6!), 115.0 (C-5"), 117.2 (C-3'), 121.8 (C-1 "), 122.3 (C-2'), 126.1 (C-5), 127.1 (C-6"), 131.2 (C-1 '), 136.2 (C-2"), 137.2 (C-4), 137.7 (C-2), 139.9 (C-3"), 144.8 <C-4'), 152.1 (C-5'), 153.1 (C-4"}; m/z (%) 463 (99) [M÷], 461 (100) [M+], 447 (46), 445 (45).
22) 1 -Methyl-5-(3"-amsno-4'>-eihoxyphenyl)-4-(3'-bromo-4',5'-dimethoxyphenyl)-imidazo!e 6f x 2HCI
Compound 6e (170 mg, 0.37 mmol) was dissolved in THF (7.5 mL). Zn powder (120 mg, 1.83 mmol) was added followed by a mixture of cone. HCI (264 μΙ_) in THF (1 mL), After stirring for 15 min at room temperature the reaction mixture was poured onto water and basified with aqueous NaHC03 to ca. pH 8. The water phase was extracted with ethyl acetate and the organic phase was dried over a2SC>4, filtered and the filtrate was concentrated in vacuum. The residue was purified by column chromatography (silica ge! 60, 10% methanol/ethyl acetate, f?f = 0.64) giving the aniline intermediate. This compound was dissolved in DCM (5 mL) and treated with 3M HCI/dioxane (1 mL). After stirring for 15 min the solvent was removed and the residue was recrystaiiized from a DCM/n-hexane mixture. Yield: 121 mg (0.22 mmol, 65%); colorless solid of m.p. >160°C (dec); vmax (ATR)/crrf1: 3369, 2936, 2829, 2540, 1632, 1547, 1492, 1411 , 1395, 1303, 1270, 1235, 1141 , 1 114, 1038, 993, 862, 818, 720; 1H NMR (300 MHz, DMSO-afe): 5 1.40 (3 H, t, 3J 6.9 Hz, Me), 3.63 (3 H, s, NMe), 3.70 (3 H, s, 4'~OMe), 3.72 (3 H, s, 5'-OMe), 4.19 (2 H, q, 3J 6.9 Hz, OCH2), 7.1 -7.3 (4 H, m, 2'-H, 6!-H, 5"-H, 6"H), 7.45 (1 H, s, 2"-H), 9.41 (1 H, s, 2-H); 13C NMR (75.5 MHz, DMSO-d6): δ 14.4 (Me), 34.0 (NMe), 56.2 (5 -OMe), 60.2 (4'-OMe), 64.6 (OCH2), 111.4 (C-6'), 1 13.7 (C-5"), 117.0 (C-3'), 1 17.5 (C-1 "), 122.4 (C-21), 124.2 (C-5), 127.6 (C-1 '), 129.3 (C-4), 135.8 (C-2), 146.0 (C-4'), 151 .7 (C-5"), 153.3 (C-4"); m/z (%) 433 (100) [M*], 431 (100) [M+], 416 (31 ), 404 (16), 402 (15), 36 (69).
23) 1 - ethyI-4-(3'-bromo-4\5!-dimethoxypheny!)-5-(3,,-fiuoro-4"-ethoxypheny!)-imi 6g x HCi
A mixture of 3-fIuoro-4-ethoxybenzaldehyde (71 mg, 0.42 mmol) and 33% MeNH2/ethanoi (260 μΙ_, 2.10 mmol) in ethanol (15 mL) was treated with AcOH (150 μΙ_, 2.63 mmo!) and refluxed for 2 h. After cooling down to room temperature, 4b (172 mg, 0.42 mmol) and K2C03 (500 mg, 3.62 mmol) were added and the reaction mixture was refluxed for 5 h. The solvent was evaporated, the residue diluted with ethyl acetate, washed with water and brine, dried over Na2S04, filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60, ethyl acetate/methanoi 9: 1 ). The resulting colorless oil was dissolved in DCM (5 mL) and treated with 3 HCI/dioxane. The reaction mixture was stirred at room temperature for 10 min, and the solvent was evaporated. The residue was recrystaliised from DCM/n-hexane. Yield: 160 mg (0.34 mmol, 81 %); colorless solid of mp 90 °C; vmax (ATR)/c?rf1 : 3391 , 31 1 1 , 2976, 2939, 2884, 2833, 2620, 1626, 1547, 1523, 1494, 1474, 142 , 1397, 1304, 1270, 1233, 1 1 15, 1039, 995, 927, 885, 851 , 809, 778, 756; 1H NMR (300 MHz, DMSO-c¾: δ 1 .37 (3 H, t, 3J 6.9 Hz), 3.71 (3 H, s), 3.73 (3 H, s), 4.19 (2 H, q, 3 J 6.9 Hz), 7.14 (1 H, d, 4 J 2.0 Hz), 7.26 (1 H, d, 4 J 2.0 Hz), 7.2-7.3 (2 H, m), 7.37 (1 H, t, 3 J 17.1 Hz), 7.51 (1 H, dd, 3 J 1 1 .9 Hz, 4 J 1 .9 Hz), 9.37 (1 H, s); 3C NMR (75.5 MHz, DMSO-cf6): δ 14.4, 33.9, 56.1 , 60.2, 64.6, 1 1 1.5, 1 15.5, 1 16.9, 1 18.6, 122.5, 124.3, 128.2, 128.9, 1 35.7, 146.1 , 148.2, 153.3; m/z (%) 436 (100) [M+], 434 (100) [M*], 421 (96), 419 (95), 340 (12), 36 (15).
24) 1 -Methyl-4-(3'-bromo-4',5'-dimethoxyphenyi)-5-(N-methyl-3"-chioroindol-5"-yl)-imidazole Gi
Analogously to the synthesis of 5a, compound 6i was obtained from the imine of W-methyl-3- chloroindol-5-carboxaldehyde (81 mg, 0.42 mmo!), 33% MeNH2/ethanol (260 pL, 2.10 mmol) and acetic acid (150 pL) in boiling ethanol (15 mL), which was treated with 4b (170 mg, 0.42 mmol) and K2C03 (500 mg, 3.62 mmol). After workup the residue was purified by column chromatography (silica gel 60). Yield: 130 mg (0.28 mmol, 67%); colorless oil; Rf = 0.63 (ethyl acetate/methanoi 95:5);vmax (ATR)/cm 1 : 31 13, 2835, 2825, 1597, 1561 , 1504, 1474, 1461 , 1418, 1396, 1373, 1312, 1256, 1240, 1 164, 1 109, 1042, 997, 971 , 907, 875, 854, 808, 728; 1H-NMR (300 MHz, CDCi3): δ 3.41 (3 H, s, 1 -NMe), 3.48 (3 H, s, 5'-OMe), 3.73 (3 H, s, 4'-OMe), 3.75 (3 H, s, 1 "-NMe), 7.01 (1 H, d, 4J 1 .9 Hz, 6'-H), 7.05 (1 H, s, 2"-H), 7.14 (1 H, dd, 3 J 8.6 Hz, 4 J 1.6 Hz, 6"-H), 7.30 (1 H, d, 4 J 1.9 Hz, 2'-H), 7.35 (1 H, d, 4 J 8.6 Hz, 7"-H), 7.52 (1 H, s, 2-H), 7.56 (1 H, d, 4 J 1.6 Hz, 4"-H); 13C-N R (75.5 MHz, CDCI3): δ 32.0 (1- NMe), 33.0 (1 "-NMe), 55.5 (5'-0Me), 60.4 (4'-0 e), 104.6 (C-3"), 109.7 (C-6'), 110.2 (C-7"), 1 17.2 (C-3'), 120.5 (C-4"), 121.4 (C-5"), 122.2 (C-2'), 125.1 (C-6"), 126.0 (C-3a"), 126.3 (C- 2"), 129.8 (C-5), 132.1 (C-1 '), 135.7 (C-7a"), 136.2 (C-4), 136.9 (C-2), 144.4 (C-4'), 152.9 (C- 5'); m/z (Ei) 461 (100) [M+], 459 (82) [M+], 446 (59), 444 (57).
25) 1 -Methyl-4-(3'-bromo-4\5 -dimethoxyphenyl)-5-(N-methyl-3"-chloroindok
hydrochioride 6i x HCI
Compound 6i (130 mg, 0.28 mmol) was dissolved in DCM (5 mL) and treated with 3M HCI/dioxane (1 mL). After stirring atroom temperature for 15min the solvent was evaporated in vacuum and the residue was recrystallised from ethanol/n-hexane. Yield: 109 mg (0.20 mmol, 73%); colorless solid; mp. 146°C; UV ( eOH) max (ε) 230 (34040);vma>! (ATR)/cm"1: 3392, 2941 , 2591 , 1622, 1592, 1544, 1490, 1464, 1406, 1317, 1263, 1239, 1146, 1115, 1042, 995, 973, 874, 853, 809; 1H-NMR (300 MHz, DMSO-d6): δ 3.61 (6 H, s, 1-NMe, 5'- OMe), 3.70 (3 H, s, 4'-OMe), 3.86 (3 H, s, 1 "-NMe), 7.14 (1 H, d, 4 J 2.1 Hz, 6'-H), 7.26 (1 H, d, 4J 2.1 Hz, 2'-H), 7.34 (1 H, dd, 3J 8.5 Hz, 4 1.6 Hz, 6"-H), 7.7-7.8 (3 H, m, 2"-H, 4"-H, 7"- H), 9.36 (1 H, s, 2-H); 13C-NMR (75.5 MHz, DMSO-d6): δ 33.0 (1-NMe), 33.9 (1 "-N e), 56.0 (5 -OMe), 60.2 (4'-OMe), 102.7 (C-3"), 1 1 1.4 (C-6'), 111.8 (C-7"), 116.9 (C-5"), 117.0 (C-3'), 120.4 (C-4"), 122.3 (C-2'), 124.5 (C-6"), 124.7 (C-3a"), 125.1 (C-7a"), 127.7 (C-5), 128.3 (C- 2"), 130.9 (C-1 '), 135.5 (C-2), 136.0 (C-4), 145.9 (C-4'), 153.2 (C-51); m/z (El) 461 (100) ijvT - 2HCI], 459 (82) [M+ - 2HCI], 446 (59), 444 (57). 26) 4-(3'i4'-dtmethoxy-5'-nitrophenyl)-5-(4"-methoxy-3I,-nitrophenyl)-oxazole 7a
Compound 4c (170 mg, 0.45 mmol), 4-methoxy-3-nitrobenzaldehyde (82 mg 0.74 mmol) and anhydrous K2C03 (590 mg, 4.3 mmol) were dissolved in DME/methanol (1 :3, 20 mL) and stirred for 2 h. The solution was concentrated in vacuum, taken up in ethyl acetate, washed with water and brine, dried over Na2S04, filtered and the filtrate was concentrated in vacuum. The residue was purified by column chromatography (siiica get 60). Yield: 140 mg (0.35 mrnoi, 78%); yellow solid of mp 155 °C; f?f - 0.30 (ethyl acetate/n-hexane 1 :1 );, vmax (ATR)/crrf1 3147, 2945, 2846, 1625, 1564, 1522, 1482, 1456, 1441 , 1342, 1278, 1267, 1256, 1184, 1162, 1126, 111 1 , 1058, 1005, 985, 849, 832; 1H NMR (300 MHz, CDCl3) δ 3.84 (3 H, s), 3.96 (3 H, s), 3.97 (3 H, s), 7.13 (1 H, d, J = 8.9 Hz), 7.38 (1 H, d, J = 2.0 Hz), 7.54 (1 H, d, J = 2.0 Hz), 7.74 (1 H, dd, J = 8.9 Hz, J = 2.3 Hz), 7.93 (1 H, s), 8.07 (1 H, d, J = 2.3 Hz); 13C NMR (75.5 MHz, CDCI3) δ 56.4, 56.7, 62.0, 1 14.1 , 114.8, 120.4, 124.0, 127,3, 132.2, 132.9, 139.7, 142.7, 144.0, 144.9, 150.1 , 153.2, 154.3; m/z (EI) 401 (34) [M+], 400 (100), 385 (12), 339 (7).
27) 4-(3\4'~dim6thoxy-5'-nitrophenyl)-5^^ 7b
Compound 4c ( 70 mg, 0.45 mmol), 4-N,A/-dimethylaminobenzaldehyde (67 mg 0.45 mmol) and anhydrous K2C03 (590 mg, 4.3 mmol) were dissolved in DME/methano! (1 :3, 20 mL) and stirred for 2 h. The soiution was concentrated in vacuum, taken up in ethyl acetate, washed with water and brine, dried over Na2S04, filtered and the filtrate was concentrated in vacuum. The residue was purified by column chromatography (silica ge! 60). Yield: 70 mg (0.19 mmol, 42%); yellow oil; Rf = 0.30 (ethyl acetate/n-hexane 1 :2); vmax (ATR)/cm~1 2941 , 2886, 16 , 1532, 1518, 1445, 1352, 1264, 1230, 1 190, 1 167, 1 104, 1053, 992, 944, 920, 870, 854, 818, 784, 771 , 733, 697; 1H NMR (300 Hz, CDC!3) 2.99 (6 H, s), 3.84 (3 H, s), 3.97 (3 H, s), 6.69 (2 H, d, J = 9.1 Hz), 7.44 (2 H, d, J = 9.1 Hz), 7.48 (1 H, d, J = 2.0 Hz), 7.65 (1 , 6, J = 2.0 Hz), 7.86 (1 H, s); 13C NMR (75.5 MHz, CDCI3) £40.1 , 56.4, 62.0, 11 1.9, 1 14.5, 1 14.8, 115.0, 128.2, 128.8, 130.1 , 141.8, 145.0, 147.8, 149.1 , 150.9, 153.9; m/z (El) 369 (100) [M+], 308 (7), 132 (12).
28) 1 -Methyl-5-(3"-benzyloxy-4"-methoxyphenyl)-4-(3',4'-dimethoxy-5'-nitrophenyl)imidazole 7c
A mixture of 3-benzoxy-4-methoxybenzaldehyde (102 mg, 0.42 mmol) and 33% MeNH2/ethanol (260 μ!_, 2.10 mmol) in ethanol (15 mL) was treated with AcOH (150 pL, 2.63 mmol) and refluxed for 2 h. After cooling down to room temperature, compound 4c (158 mg, 0.42 mmol) dissolved in DME (5 mL) and K2C03 (500 mg, 3.62 mmol) were added and the reaction mixture was refluxed for 3 h. The solvent was evaporated, the residue diluted with ethyl acetate, washed with water and brine, dried over Na2S0 , filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60). Yield: 130 mg (0.27 mmol, 64%); red oil; Rf = 0.58 (ethyl acetate/methanol 95:5); vmax (ATR)/crrT1 2939, 2837, 1529, 1509, 1454, 1358, 1248, 1169, 1136, 1 110, 1060, 1021 , 993, 919, 858, 814, 764, 729, 696; 1H NMR (300 MHz, CDCI3) £3.25 (3 H, s), 3.61 (3 H, s), 3.86 (3 H, s), 3.89 (3 H, s), 5.09 (2 H, s), 6.74 (1 H, d, J = 2.0 Hz), 6.85 (1 H, dd, J = 8.2 Hz, J = 1.9 Hz), 6.95 (1 H, d, J = 1.9 Hz), 7.1-7.3 (6 H, m), 7.36 (1 H, d, J = 2.0 Hz), 7.45 (1 H, s); 13C NMR (75.5 MHz, CDCl3) 31.7, 55.8, 55.9, 61.7, 71.0, 1 12.1 , 113.0, 1 13.4, 1 16.3, 121.6, 123.7, 127.0, 127.8, 128.4, 129.4, 130.9, 135.4, 136.4, 137.2, 140.5, 144.6, 148.2, 150.4, 153.3; m/z (El) 475 (62), 458 (26), 384 (21 ), 337 (22), 91 (100), 65 (36). 29) 1 -Methyt-4-(3',4'-dimethoxy-5'-nitrophenyl)-5-(3"-f luoro-4"-methoxyphenyl)-imidazoie 7d
A mixture of 3-fluoro-4-methoxybenzaldehyde (77 mg, 0.42 mmol) and 33% MeNH2/eihanol (260 μ!_, 2.10 mmol) in ethanol (15 mL) was treated with AcOH (150 μΐ_, 2.63 mmol) and refluxed for 2 h. After cooling down to room temperature, compound 4c (158 mg, 0.42 mmoi) dissolved in DME (5 mL) and K2C03 (500 mg, 3.62 mmol) were added and the reaction mixture was refluxed for 3 h. The solvent was evaporated, the residue diluted with ethyi acetate, washed with water and brine, dried over Na2S04, filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60). Yield: 120 mg (0.31 mmoi, 74%); red oil; f = 0.45 (ethyl acetate/methanol 95:5); vmax (ATR)/cm"1 2941 , 2841 , 1530, 1511 , 1462, 1357, 1300, 1267, 1238, 1131 , 1110, 1021 , 993, 920, 867, 855, 817, 761 , 730, 657; 1H N R (300 MHz, CDCI3) £ 3.43 (3 H, s), 3.69 (3 H, s), 3.86 (3 H, s), 3.89 (3 H, s), 7.0-7.1 (3 H, m), 7.29 (1 H, d, J = 2.0 Hz), 7.30 (1 H, d, J = 2.0 Hz), 7.50 (1 H, s); 13C NMR (75.5 MHz, CDCI3) 32.0, 56.0, 56.1 , 61.8, 1 13.2, 1 13.7, 1 13.9, 1 17.9, 118.2, 121.9, 122.0, 126.9, 128.2, 130.7, 135.9, 137.5, 140.7, 144.7, 148.3, 148.5, 150.7, 153.5, 154.0; m/z (El) 387 (100) [M+], 372 (34), 212 (20).
30) N-Methyl-3-chloroindol-5-carbaldehyde 7e' A/-Methylindol-5-carbaldehyde (400 mg, 2.5 mmol) was dissolved in dry acetonitrile (10 mL) and treated with /V-chiorosuccinimide (400 mg, 3.02 mmoi), whereupon the solution turned red. The reaction mixture was stirred at room temperature for 20 h. The solvent was removed in vacuum and the residue was purified by column chromatography (silica gel 60). Yield: 300 mg (1.55 mmol, 62%); colorless soiSd of mp 109 °C; f?f = 0.27 (ethyl acetate/n-hexane 1 :4);vmax (ATR)/cm"1 3103, 2845, 2751 , 1680, 1602, 1454, 1414, 1362, 1343, 1274, 1238, 1196, 1 160, 1134, 1111 , 981 , 894, 796, 717; 1H NMR (300 MHz, CDCI3) 3.75 (3 H, s), 7.07 (1 H, s), 7.32 (1 H, d, J = 8.6 Hz), 7.76 (1 H, dd, J = 8.6 Hz, J = 1.5 Hz), 8.07 (1 H, d, J = 1.5 Hz), 10.01 (1 H, s); 13C NMR (75.5 MHz, CDCl3) £ 32.9, 106.3, 109.9, 122.0, 123.3, 125.2, 126.9, 129.2, 138.5, 191.7; m/z (El) 193 (100) [M+], 164 (57), 128 (43), 101 (48), 87 (28).
31 ) 1-MethyI-4-(3',4'-dimethoxy-5'-nitrophenyl)-5-(N-methyl-3"-chioroindol-5"-yl)-imidazoie 7e
A mixture of /V-methyl-5-chloroindol-3-carbaldehyde (81 mg, 0.42 mmol) and 33% MeNH2/ethanol (260 pL, 2.10 mmol) in ethanol (15 mL) was treated with AcOH (150 pL, 2.63 mmol) and refluxed for 2 h. After cooling down to room temperature, compound 4c (170 mg, 0.43 mmoi) and 2C03 (500 mg, 3.62 mmoi) were added and the reaction mixture was refluxed for 3 h. The solvent was evaporated, the residue diluted with ethyl acetate, washed with water and brine, dried over Na2S04, filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60). Yield: 120 mg (0.28 mmo!, 67%); red oil; f = 0.52 (ethyl acetate/methanol 95:5); vmax (ATR)/crn 1 3118, 2940, 2830, 1528, 1507, 1478, 1357, 1263, 1239, 1 1 1 1 , 1060, 993, 863, 804, 730; 1H NMR (300 MHz, CDCI3) 3.42 (3 H, s), 3.57 (3 H, s), 3.76 (3 H, s), 3.84 (3 H, s), 7.07 (1 H, s), 7.14 (1 H, dd, J = 8.5 Hz, J = 1.6 Hz), 7.3-7.4 (3 H, m), 7.54 (1 H, s), 7.55 (1 H, d, J = 1.6 Hz); 13C NMR (75.5 MHz, CDCI3) £32.0, 33.0, 55.9, 61.7, 104.6, 1 10.5, 113.0, 1 13.5, 120.4, 120.9, 124.8, 126.1 , 126.5, 130.5, 131.2, 135.5, 135.8, 137.1 , 140.4, 144.6, 153.3; m/z (El) 428 (38) [M+], 426 (100) [M+], 41 1 (26).
32) 4-(3 -Amino-4',5'-dimethoxyphenyi)-5-(3"-amino-4"-methoxyphenyl)-oxazole 8a
Compound 7a (120 mg, 0.30 mmol) was suspended in methanol (20 mL) and treated with ammonium formate (590 mg, 9.37 mmol) and Pd/C (5%, 180 mg). The suspension was refluxed for 2 h and after cooling to room temperature the mixture was filtered over celite, the filtrate was concentrated in vacuum and the residue was purified by column chromatography (silica gel 60). Yield: 70 mg (0.21 mmol, 70%); light brown solid of mp 63 °C; Rf = 0.55 (ethyl acetate); UV (MeOH) max (ε) 285 (7720); vmax (ATR)/cm"1 3457, 3361 , 2935, 2834, 1613, 1587, 1515, 1428, 1378, 1222, 1175, 1137, 1 105, 999, 843, 802, 760; 1H NMR (300 MHz, CDCI3) £ 3.73 (3 H, s), 3.80 (3 H, s), 3.82 (3 H, s), 3.9-4.0 (4 H, broad s), 6.6-6.8 (3 H, m), 6.9-7.0 (2 H, m), 7.83 (1 H, s); 13C NMR (75.5 MHz, CDCI3) £55.4, 55.6, 59.8, 102.1 , 108.2, 1 10.1 , 113.2, 117.6, 121.5, 128.1 , 133.2, 135.6, 136.1 , 140.4, 145.7, 147.6, 148.9, 152.7; m/z (Ei) 343 (45), 341 (66) [M*], 326 (46), 298 (22), 197 (63), 91 (100), 57 (75).
33) 4-(3'-Amino-4',5'-dimethoxyphenyl)-5-(3"-amino-4!'-methoxyphenyl)-oxazole bis(hydrochloride) 8a x 2HCI
Compound 8a (60 mg, 0.18 mmol) was dissolved in DCM (5 mL) and treated with 3M HCI/dioxane (1 mL). The reaction mixture was stirred at room temperature for 15 min and the formed coiorless precipitate was collected, washed with DCM and dried in vacuum. Yield: 28 mg (0.07 mmol, 39%); colorless solid of mp 227 °C; UV (MeOH) (ε) 292 (11960); vmax (ATR)/cm"1 2837, 2553, 1633, 1567, 1518, 1495, 1376, 1270, 1142, 1 102, 1052, 996, 934, 869, 839, 817, 760, 722; 1H NMR (300 MHz, DMSO-d6) £3.74 (3 H, s), 3.85 (3 H, s), 3.92 (3 H, s), 7.10 (1 H, s), 7.19 (1 H, s), 7.25 (1 H, d, J = 8.7 Hz), 7.4-7.5 (1 H, m), 7.56 (1 H, d, J = 2.1 Hz), 8.52 (1 H, s); 13C NMR (75.5 MHz, DMSO-d6) £ 55.9, 56.2, 60.7, 108.9, 1 12.6, 1 13.1 , 120.2, 120.6, 124.8, 125.4, 127.5, 128.9, 132.4, 140.3, 144.3, 151.2, 151.6, 152.7; m/z (EI) 341 (100) [M+ - 2HCI], 376 (72), 298 (25), 255 (12), 212 (7), 184 (7), 135 (8), 78 (11 ), 65 (6). 34) 4-(3'-Amino-4\5!-dimethoxypheny1^ 8b
Compound 7b (100 mg, 0.27 mmoi) was suspended in methanol (20 mL) and treated with ammonium formate (590 mg, 9.37 mmoi) and Pd/C (5%, 180 mg). The suspension was refiuxed for 2 h and after cooiing to room temperature the mixture was ftitered over ceiite, the filtrate was concentrated in vacuum and the residue was purified by column chromatography (silica gel 60). Yield: 60 mg (0.18 mmoi, 67%); colorless oil; Rf = 0.77 (ethyl acetate/methanol 95:5); vmax (ATR)/cnY1 3427, 3295, 3189, 2927, 2820, 1617, 1583, 1523, 1512, 1445, 1426, 1382, 1363, 1327, 1278, 1227, 1 190, 1 146, 1 105, 1057, 1005, 949, 842, 81 ; 1H NMR (300 MHz, CDCi3) 2.96 (6 H, s), 3.76 (3 H, s), 3.82 (3 H, s), 6.6-6.8 (4 H, m), 7.49 (2 H, d, J = 9.1 Hz), 7.82 (1 H, s); 13C NMR (75.5 MHz, CDCI3) £40.1 , 55.7, 59.9, 102.0, 108.0, 1 1 1.8, 116.4, 128.0, 128.5, 132.2, 135.5, 140.4, 146.3, 148.6, 150.4, 152.8; m/z (El) 339 (82) [M+], 324 (70), 296 (25), 253 (44), 159 (41 ), 148 (100), 133 (59), 1 19 (84), 78 (67), 42 (54).
35) 4-(3'-Amino-4',5'-dimethoxyphenyl)-5-(4"-N,N-dimethylaminophenyl)-oxazofe bis(hydrochloride) 8b x 2HCI
Compound 8b (58 mg, 0.17 mmoi) was dissolved in DCM (5 mL) and treated with 3M HCl/dioxane (1 mL). The reaction mixture was stirred at room temperature for 15 min, the solvent was removed and the formed colorless solid was recrystailized from DCM/n-hexane. Yield: 70 mg (0.17 mmoi, 100%); yellow solid of mp 74 °C; UV (MeOH) λ™* (ε) 327 (18200), 259 (16360); vmax (ATR)/cm"1 3382, 2941 , 2840, 2548, 1572, 1541 , 1512, 1496, 1418, 1378, 1276, 1251 , 1104, 1062, 991 , 935, 870, 850; 1H NMR (300 MHz, DMSO-d6) £2.99 (6 H, s), 3.74 (3 H, s), 3.87 (3 H, s), 6.9-7.1 (2 H, m), 7.22 (1 H, d, J = 1.9 Hz), 7.28 (1 H, d, J = 1.9 Hz), 7.49 (2 H, d, J = 8.9 Hz), 8.46 (1 H, s); 13C NMR (75.5 MHz, DMSO-d6) £ 40.7, 55.9, 61.0, 1 10.4, 1 13.8, 1 13.9, 126.7, 128.0, 128.2, 130.9, 141.0, 145.9, 149.4, 150.7, 152.6; m/z (El) 339 (82) [M+ - 2HCI], 324 (70), 296 (25), 253 (44), 159 (41 ), 148 (100), 133 (59), 1 19 (84), 78 (67), 42 (54).
36) 1-Methyl~4-(3'-amino-4',5'-dimethoxyphenyi)-5-(3"-hydroxy-4-methoxyphenyl)-imidazole bis(hydrochioride) 8c x 2HCI Compound 7c (120 mg, 0.31 mmo!) was dissolved in methanol (20 mL) and treated with ammonium formate (590 mg, 9.37 mmol) and Pd/C (5%, 180 mg). The suspension was refluxed for 2 h and after cooling to room temperature the mixture was filtered over ceiite, the filtrate was concentrated in vacuum and the residue was purified by column chromatography giving 8c (siiica gel 60, ethyl acetate/methanol 95:5, Rf = 0.25). Compound 8c was dissolved in DCM (5 mL) and treated with 3M HCI/dioxane (1 mL). The reaction mixture was stirred at room temperature for 15 min. The solvent was removed and the residue freed from dtoxane by repeated azeotropic distillation with DCM. The remaining solid was recrystallized from ethanol/n-hexane. Yield: 70 mg (0.16 mmol, 53%); colorless solid of mp 218 °C (dec); UV (MeOH) (ε) 275 (1 1300); vmax (ATR)/cm"1 3032, 2967, 2522, 1620, 1590, 1534, 1508, 1432, 1328, 1292, 1251 , 121 1 , 1 135, 1 109, 1051 , 1026, 992, 855, 813, 766; 1H NMR (300 MHz, DMSO-d6) £ 3.61 (3 H, s), 3.73 (3 H, s), 3.77 (3 H, s), 3.83 (3 H, s), 6.75 (1 H, d, J 1 .7 Hz), 6.8-7.0 (3 H, m), 7.09 (1 H, d, 3J 8.5 Hz), 9.33 (1 H, s); 1 C NMR (75.5 MHz, DMSO-d6):
33.9, 55.6, 55.9, 60.3, 109.5, 1 12.7, 1 17.5, 122.1 , 122.8, 128.2, 129.9, 135.2, 147.1 , 149.3, 152.6; m/z (El) 356 (33), 355 (85) [M+ - 2HCI], 341 (42), 340 (100).
37) 1 -Methyl-4-(3'-amino-4',5'-dimethoxyphenyl)-5-(3"-fluoro-4-methoxyphenyl)-imidazole 8d
Compound 7d (120 mg, 0.31 mmol) was dissolved in methanol (20 mL) and treated with ammonium formate (590 mg, 9.37 mmol) and Pd/C (5%, 180 mg). The suspension was refluxed for 2 h and after cooling to room temperature the mixture was filtered over ceiite, the filtrate was concentrated in vacuum and the residue was purified by column chromatography (silica gel 60). Yield: 80 mg (0.25 mmol, 81 %); colorless gum; Rf = 0.33 (ethyl acetate/methanol 95:5); vmax (ATR)/cm_ 3448, 3364, 2940, 2835, 1610, 1561 , 1 517, 1500, 1463, 1442, 1394, 1301 , 1268, 1237, 1208, 1 132, 1 107, 1049, 1024, 1002, 892, 877, 848, 817, 761 , 658; H NMR (300 MHz, CDCI3) £ 3.38 (3 H, s), 3.5-3.6 (2 H, broad s), 3.59 (3 H, s), 3.72 (3 H, s), 3.87 (3 H, s), 6.47 (1 H, d, J = 1 .9 Hz), 6.48 (1 H, d, J = 1 .9 Hz), 6.9-7.1 (3 H, m), 7.45 (1 H, s); 3C NMR (75.5 MHz, CDCl3) £ 31 .9, 55.2, 56.1 , 59.7, 100.1 , 106.9, 1 13.5, 1 18.2, 1 18.4, 123.1 , 123.2, 126.9, 127.0, 127.1 , 130.3, 1 34.6, 137.0, 138.1 , 140.0, 147.7, 147.9, 150.5, 152.4, 153.7; m/z (El) 358 (57), 357 (93) [M+], 343 (75), 342 (100), 282 (47), 243 (51 ), 229 (50), 200 (42), 158 (34), 42 (72).
38) 1 -Methyl-4-(3'-amino-4',5'-dimethoxyphenyl)-5-(3"-fluoro-4-methoxyphenyl)-imidazole bis(hydrochloride) 8d x 2HC1
Compound 8d (80 mg, 0.25 mmol) was dissolved in DCM (5 mL) and treated with 3M HCI/dioxane (1 mL). The reaction mixture was stirred at room temperature for 15 min. The solvent was removed and the residue freed from dioxane by repeated azeotropic distillation with DCM. The remaining solid was recrystaliized from ethanol/n-hexane. Yield: 90 mg (0.21 mmoi, 84%); colorless solid of mp 214-217 °C; UV (MeOH) max (ε) 270 (1 1 100); vmax (ATR)/cm"1 2969, 2323, 1625, 1555, 1526, 1494, 1473, 1422, 1341 , 1305, 1278, 1242, 1 178, 1 131 , 1 112, 1061 , 1025, 974, 858, 825, 763; 1H NMR (300 MHz, DMSO-d6) δ 3.63 (3 H, s), 3.77 (3 H, s), 3.78 (3 H, s), 3.92 (3 H, s), 6.66 (1 H, s), 7.05 (1 H, s), 7.2- 7.3 (1 H, m), 7.35 (1 H, t, J = 17.3 Hz), 7.47 (1 H, d, J - 12.0 Hz), 9.37 (1 H, s); 13C NMR (75.5 MHz, DMSO-d6) 34.0, 55.0, 56.2, 60.3, 1 1 1.4, 1 14.7, 1 17.6, 1 17.7, 1 18.2, 118.5, 122.6, 128.1 , 128.5, 129.0, 135.4, 140.0, 148.7, 148.8, 149.8, 152.6, 153.0; m/z (El) 358 (63), 357 (96) [M+ - 2HCI], 343 (79), 342 (100), 284 (37).
39) 1 -lvlethyl-4-(3,-amino-4',5,-dimethoxyphenyi)-5-(N-methyS-3"-chloroindoi-5"-yl)-imidazole 8e Compound 7e (120 mg, 0.28 mmol) was dissolved in THF (7.5 mL) and reduced by adding Zn powder (90 mg, 1.39 mmo!) and cone. HCI (200 μΙ_) in THF (1 mL). After workup the residue was purified by column chromatography (silica ge! 60). Yield: 70 mg (0.18 mmol, 64%); colorless oil; Rf = 0.44 (ethyl acetate/methanol 95:5); vmax (ATR)/cm"1 3359, 31 13, 2934, 2825, 1613, 1593, 151 1 , 1480, 1447, 1423, 1396, 1336, 1239, 1224, 1 139, 1109, 1000, 974, 907, 864, 803, 726; 1H NMR (300 MHz, CDCl3) £3.39 (3 H, s), 3.52 (3 H, s), 3.70 (3 H, s), 3.74 (3 H, s), 6.50 (1 H, d, J = 1.9 Hz), 6.58 (1 H, d, J = 1.9 Hz), 7.05 (1 H, s), 7.16 (1 H, dd, J = 8.5 Hz, J = 1 .6 Hz), 7.32 (1 H, d, J = 8.5 Hz), 7.50 (1 H, s), 7.58 (1 H, d, J = 1.6 Hz); 13C NMR (75.5 MHz, CDCl3) 532.0, 33.0, 55.3, 59.7, 101.0, 104.6, 106.8, 1 10.1 , 120.6, 122.1 , 125.5, 125.9, 126.1 , 129.1 , 130.8, 134.5, 135.6, 136.6, 137.6, 139.8, 152.4; m/z (El) 398 (19) [M+], 396 (56) [M+], 383 (37), 381 (100), 321 ( 1 ), 282 (12), 42 (23).
40) 1 -Methyl-4-(3I-amino-4',5'-dimethoxyphenyi)-5-(N-methyl-3"-chioroindol-5"-yl)-imidazole tris(hydrochloride) 8e x 2HCI Compound 8e (70 mg, 0.18 mmol) was dissolved in DCM (5 mL) and treated with 3M HCI/dioxane (1 mL). The reaction mixture was stirred at room temperature for 15 min. The solvent was removed and the residue freed from dioxane by repeated azeotropic distillation with DCM. The remaining solid was recrystaliized from ethanol/n-hexane. Yield: 62 mg (0.12 mmol, 68%); brown solid of mp >300 °C (dec); UV (MeOH) Xmax (ε) 224 (30100); vmax (ATR)/cm"1 3356, 2830, 2570, 1623, 1554, 1498, 1456, 1423, 1408, 1332, 1284, 1272, 1237, 1 145, 1 12, 1064, 991 , 973, 857, 804; 1H NMR (300 MHz, DMSO-d6) £3.61 (3 H, s), 3.63 (3 H, s), 3.73 (3 H, s), 3.89 (3 H, s), 6.52 (1 H, s), 6.79 (1 H, s), 7.30 (1 H, dd, J = 8.5 Hz, J = 1.6 Hz), 7.6-7.8 (3 H, m), 9.35 (1 H, s); 13C NMR (75.5 MHz, DMSO-d6) S 33.0, 34.0, 55.7, 60.0, 102.7, 1 1 1.7, 1 17.0, 120.3, 122.8, 124.6, 125.1 , 128.0, 128.9, 130.4, 135.1 , 136.0, 152.5; m/z (Ei) 396 (42) [ +- 2HCI], 383 (45), 381 (78), 331 (21 ), 160 (100), 57 (59), 41 (65). 41 ) (3-Fluoro-4-methoxyphenyl)-(p-to!uenesutfonyl)methylisocyanide 4d
A mixture of 3-fluoro-4-methoxybenzaldehyde (3.9 g, 22.94 mmoi), p-toiuenesulfinic acid (3.0 g, 19.29 mmoi), camphorsulfonic acid (1 10 mg) and formamide (10 mL) was stirred at 65 °C for 16 h. After cooling in an ice bath the mixture was treated with water and the resulting precipitate was separated, washed with a little methanol and dried in vacuum to ieave the N- substituted formamide (1.9 g, 5.64 mmol, 30%). This compound was dissolved in dry DME (50 mL), cooled to -5 °C and treated with POC!3 (1.7 mL) and Et3N (4.15 mL). The reaction mixture was stirred at -5 °C for 2 h. The resulting suspension was poured on ice water and extracted with ethyl acetate. The organic phase was washed with aqueous NaHC03 and brine, dried over Na2S04, filtered and the filtrate was concentrated in vacuum. The residue thus obtained was purified by column chromatography (silica ge! 60). Yield: 560 mg (1.76 mmol, 31 %); Rf = 0.41 (ethyl acetate/n-hexane, 1 :2); brown solid of mp 86-90 °C (dec.);vmax (ATR)/cm"1 2947, 2845, 2134, 1620, 1595, 1518, 1441 , 1324, 1304, 1273, 1225, 1 148, 1 126, 1082, 1018, 81 1 , 761 , 664; 1H NMR (300 MHz, CDCI3) J2.46 (3 H, s), 3.90 (3 H, s), 5.50 (1 H, s), 6.05 (1 H, t, JHF = 16.8 Hz), 7.0-7.2 (2 H, m), 7.33 (2 H, d, J = 7.4 Hz), 7.63 (2 H, d, J = 7.4 Hz); 13C NMR (75.5 MHz, CDCI3) 21.8, 56.3, 75.6, 1 13.1 , 1 13.2, 116.1 , 116.3, 1 18.8, 118.9, 124.9, 125.0, 129.9, 130.5, 146.8, 149.7, 149.8, 150.3, 153.6, 166.3; m/z (El) 344 (12) [M+3, 319 (5), 278 (34), 246 (22), 164 (100), 155 (61 ), 139 (74), 102 (33), 91 (99), 65 (44). 42) 1-Methyl-5-(3-bromo-4,5-dimethoxyphenyi)-4-(3-fluoro-4-methoxyphenyl)imidazole x HCI 9a
A mixture of 3-bromo-4,5-dimethoxybenzaldehyde (86 mg, 0.35 mmol) and 33% MeNH2/ethanol (220 pL, 1.78 mmol) in ethanol (15 mL) was treated with AcOH (125 pL, 2.19 mmo!) and refluxed for 2 h. After cooling to room temperature, 4d (110 mg, 0.35 mmoi) and K2C03 (500 mg, 3.62 mmoi) were added and the reaction mixture was refluxed for 5 h. The solvent was evaporated, the residue was diluted with ethyl acetate, washed with water, dried over Na2S04, filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60, ethyl acetate, Rf = 0.38) to Ieave the imidazole as a colorless oil. This was dissolved in CH2CI2 (5 mL) and treated with 3 M HCI in dioxane (1 mL). After stirring for 5 min the solvent was evaporated and the residue crystallized from CH2CI2/hexane. Yield; 60 mg (37%); off-white solid of mp 85-86 °C;vmax (ATR)/cm^ 3011 , 2940, 2841 , 2605, 1627, 1590, 1552, 1520, 1499, 1464, 1443, 1421 , 1400, 1307, 1275, 1239, 1208, 1181 , 1147, 1 136, 1 105, 1023, 994, 894, 864, 809, 762, 730, 694; 1H NMR (300 MHz, DMSO-d6) £3.65 (3 H, s), 3.82 (3 H, s), 3.83 (3 H, s), 3.84 (3 H, s), 7.1-7.4 (5 H, m), 9.23 (1 H, s); 13C NMR (75.5 MHz, DMSO-d6) 33.9, 56.1 , 56.5, 60.3, 114.3, 114.5, 1 14.8, 1 15.3, 1 17.2, 123.1 , 123.9, 126.5, 127.8, 135.7, 147.1 , 149.5, 152.7, 153.8; m/z (EI) 422 (92) [M+, free base], 420 (92) [M+], 407 (24), 405 (23).
43) 1 -Methyl-4-(3-f!uoro-4-meihoxyphenyl)-5-(3,5-dibromo-4-methoxyphenyl)-imidazoje x HCi 9b
A mixture of 3,5-dibromo-4-methoxybenzaidehyde (103 mg, 0.35 mrnoi) and 33% MeNH2/ethanol (220 μΙ_, 1.78 mrnoi) in ethano! (15 mL) was treated with AcOH (125 μΙ_, 2.19 mmol) and refluxed for 2 h. After cooling to room temperature, 4d (110 mg, 0.35 mmol) and K2C03 (500 mg, 3.62 mmol) were added and the reaction mixture was refluxed for 5 h. The solvent was evaporated, the residue was diluted with ethyi acetate, washed with water, dried over Na2S04, filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60, ethyl acetate/methanol 9:1 , Rf = 0.6) giving the imidazole as a colorless oil. This oil was dissolved in CH2CI2 (5 mL) and treated with 3 M HCI in dioxane (1 mL). After stirring for 5 min the soivent was evaporated and the residue was crystallized from CH2CI2/hexane. Yield: 90 mg (51 %); colorless solid of mp 125-130 °C;vmax (ATR)/cm"1 3380, 3123, 301 1 , 2940, 2841 , 2610, 1628, 1585, 1551 , 1522, 1494, 1463, 1442, 1411 , 1307, 1278, 1258, 1208, 1163, 1137, 1 106, 1021 , 988, 877, 814, 754, 723; 1H NMR (300 MHz, DMSO-dtj) £ 3.65 (3 H, s), 3.84 (3 H, s), 3.89 (3 H, s), 7.0-7.1 (1 H, m), 7.23 (1 H, t, JHF = 17.7 Hz), 7.32 (1 H, dd, J = 12.5 Hz, 2.2 Hz), 7.86 (2 H, s), 9.19 (1 H, s); 13C NMR (75.5 MHz, DMSO-d6) £ 33.9, 56.1 , 60.6, 114.3, 1 14.7, 115.0, 118.2, 124.1 , 125.2, 126.2, 135.2, 136.1 , 147.8, 149.5, 152.8, 154.9; m/z (El) 472 (52) [M+, free base], 470 (100) [M+, free base], 468 (28) [M+, free base], 455 (19), 330 (19), 36 (40).
44) 1 -Methyl-4-(3-fluoro-4-methoxyphenyl)-5-(3,5-ditodo-4-methoxyphenyl)-imidazole x HC! 9c
A mixture of 3,5-diodo-4-methoxybenzaldehyde (160 mg, 0.41 mmol) and 33% MeNH2/ethanol (260 pL, 2.10 mmol) In ethanol (15 mL) was treated with AcOH (150 pL, 2.63 mmol) and refluxed for 2 h. After cooling to room temperature, 4d (150 mg, 0.47 mmol) and K2C03 (500 mg, 3.62 mrnoi) were added and the reaction mixture was refluxed for 5 h. The solvent was evaporated, the residue was diluted with ethyl acetate, washed with water, dried over Na2S04, filtered and concentrated in vacuum. The residue was purified by column chromatography (silica gel 60, ethyl acetate, f = 0.48) affording the imidazole as a colorless oil. This oil was dissolved in CH2CI2 (5 mL) and treated with 3 M HCI in dioxane (1 mL). After stirring for 5 min the solvent was evaporated and the residue crystallised from CH2CI2/hexane. Yield: 60 mg (25%); off-white solid of mp 207-210 °C (dec.);vmax (ATR)/cm~1 31 1 1 , 3055, 3003, 2928, 2841 , 2627, 1633, 1571 , 1549, 1519, 1491 , 1469, 1443, 1393, 1337, 1309, 1280, 1258, 1208, 1183, 1149, 1106, 1068, 1016, 991 , 894, 875, 863, 814, 767, 741 , 696; H N R (300 MHz, DMSO-d6) 3.62 (3 H, s), 3.83 (3 H, s), 3.84 (3 H, s), 7.0-7.1 (1 H, m), 7.2-7.3 (2 H, m), 7.98 (2 H, s), 9.1 1 (1 H, s); 13C NMR (75.5 MHz, DMSO-d6) £34.4, 56.8, 60.9, 92.8, 1 14.9, 1 5.0, 1 15.2, 1 15.5, 124.4, 124.5, 126.6, 136.6, 142.3, 148.1 , 150.0, 160.4; m/z (El) 564 (100) [M+, free base], 549 (33).
Example 3: Biological assays 1-Methyi-5-(3-amino-4-methoxyphenyl)-4-(3-chioro-4,5-dtmethoxyphenyl)-imidazole ("5b") as a representative compound of the present invention has been subjected to in vitro and in vivo assays, as described in the following.
For the biotests compound 5b was dissolved in physiological saline solution. Concentrations as high as 10 mM could be achieved thus demonstrating the good solubility. Customarily, in vivo applications to animals require only concentrations up to 3 mM. In order to check the chemical stability of such solutions of 5b they were stored for one month and subsequently used in in vitro tests alongside freshly prepared solutions of the same concentrations of 5b. Analysis of tubulin polymerization was performed using the Tubulin Polymerization Assay Kit (Cytoskeieton, USA) according to manufacturer's instructions. The assay is fluorescence- based and tubulin polymerization was followed by measuring RFU (relative fluorescence units) on the SpectraFluorPlus (Tecan, Switzerland) using the following filters: excitation 360 nm, emission 465 nm.
The cytotoxic activity of 5b was established by standard colorimetric tests (SRB- and MTT- assays) against various tumor cell lines of different entities. For the SRB cytotoxicity assay, dose-response curves of the testicular germ cell tumor cell lines exposed to drug concentrations of 0.001-10 μΜ were established using the sulforhodamine-B (SRB) microculture colorimetric assay (Papazisis et al. Optimization of the sulforhodamine B colorimetric assay. J, Immunol. Methods 1997, 208, 151 -158) and performed as described in: Muller et al. Failure of activation of caspase-9 induces a higher threshold for apoptosts and cispiatirt resistance in testicular cancer. Cancer Res. 2003, 63, 513-521 , Briefly, cells were seeded into 96-weil plates on day 0, at cell densities previously determined to ensure exponential cell growth during the period of the experiment. On day 1 , ceils were treated with the drugs dissolved in medium to give the appropriate concentrations for indicated times and the percentage of surviving cells relative to untreated controls was determined on day 5.
The assays for inhibition of the polymerization of tubulin and for the cytotoxicities revealed high, unattenuated activity and efficacy of 5b, as shown in Figures 1 and 2. Unlike cisplatin, compound 5b showed similar in vitro activity against the two germ cell tumor cell lines H12.1 and 141 1 HP with IC50{96 h) = 30-50 nM, i.e., it overcame the chemoresistance of the 141 1 HP ceiis (Figure 3A). This suggests that compound 5b operates by a differential mechanism of action, initiating growth inhibition and ceil death aiso in multidrug resistant tumor cells. Comparable results can be obtained, e.g., with compound 6b according to the invention (Figure 3B).
In vivo antitumor activity and tolerability of compounds 5b and 6b of the invention were studied in a nude mouse xenograft model of the resistant germ cell tumor cell line 141 1 HP employing Athymic Nude-Fox n1 nu/nu mice (Harlan und Winkeimann, Borchen, Germany).
Prior to testing compound 5b, healthy animals of this type were treated with an ascending range of doses of 5b in order to establish a toxicity profile. The compound was very well tolerated by the mice even at relatively high concentrations. Suitable dosages were then administered In further tests in a nude mouse xenograft model of a pair of germ cell tumor cell lines, the chemoresistant cell line 1411 HP and the chemosensitive cell line H12.1. These ceil lines retain their individual in vitro chemosensitivities to cisplatin when grown as xenograft tumors and so may serve as a model of the clinical situation of resistance to chemotherapy (Mueller et al. Cancer Res. 2003; Mueller et al. Tumor Biol. 2006). While treatment of H12.1 xenograft bearing mice with cisplatin led to complete regressions, 141 1 HP xenografts suffered only a transient growth inhibition followed by renewed tumor progress.
The tests were carried out as follows. The mice were kept under pathogen-free conditions, fed on an autoclaved standard diet and given free access to sterilized water. Each of five mice were administered a 150 μ1_ phosphate buffered saline suspension of 10 million 141 1 HP cells into the left flank to generate subcutaneous xenograft tumors. After 4 weeks one group of two mice bearing 141 1 HP xenografts with a volume of ca. 1 cm3 were injected i.p. with a single dose of 30 mg/kg body weight of 5b or 6b, respectively. Two mice in the second group with xenografts of a volume of ca. 2.5 cm3 were injected i.p. with 20 mg/kg body weight of 5b on two consecutive days. Tumor volumes were calculated by caliper measurement using the formula a2 * b * 0.5 with a being the short and b the Song dimension. Body weight was assessed twice weekly and daily while under therapy.
After the two mice bearing 141 1 HP xenografts with a volume of about 1 cm3 were treated with a single dose of 30 mg/kg body weight of 5b or 6b, in either case a strong intra-tumoral hemorrhage became visible yet after 24 h as a red-b!ue to brown coloring, as shown in Figure 5 for treatment with 5b. Also visible was a transient swelling of the tumors, eventually followed by regression and slow regrowth, as shown in Figure 4 A. Both mice tolerated this treatment very well. A prolonged period of tumor shrinkage was achieved in the two mice bearing 1411 HP xenografts of about 2.5 cm3 volume by administration of 20 mg/kg body weight of 5b on two consecutive days. The resulting dramatic tumor regressions, leading even to a stabilization in one case, are shown in Figures 4 B/C. The other xenograft regrew and was given two further double doses of 5b on days 16/17 and 35/36. As shown in Figure 4 C, regressions and prolonged periods of stabilization following each of the three applications were achieved. Notably, even the third course of treatment was well tolerated and the mouse had regained its original body weight by this time. These data demonstrate the great potential of the compounds 5b and 6b for an efficient curative treatment of resistant tumors.
In the mouse model anti-tumor activity of 5b thus became apparent by a dramatic regression of the resistant tumors (Figure 4). The strong vascular disrupting effect selectively affecting the tumoral vasculature was demonstrated and visualized by a distinct intra-tumoral hemorrhage occurring 24 h after administration of the compound 5b (Figure 5). In addition, a single dose application of compound 5b was sufficient to stabilize the tumor volume for about two weeks. Furthermore, a tumor regression trial using dual-dose i. p. application of 2x20mg/kg body weight of compound 6b in 141 1 HP germ cell tumor xenograft showed high antitumor activity to a similar extent as compound 6b, as also shown in Figure 10.
From these findings it can be concluded that the compounds of formula (I), such as, e.g., compound 5b or 6b, represent a class of effectives suitable for the treatment of multi- resistant tumors. Their hallmark is the synergistic combination of high cancer ce!l-specific cytotoxicity based upon a genuine mechanism, a tumor-selective vascular disrupting effect, favourable pharmacological properties, and excellent tolerance in vivo.
The compounds 5f, 6b and 6f were further tested for vascular disrupting effect in the 141 1 HP xenograft model, and the compounds 5b, 5f, 6b and 6f were additionally tested in a second tumor model of A2780 ovarian carcinoma xenografts, showtng vascular disrupting activity of all compounds in both models (Figure 1 1 ), which suggests a general activity of the compounds of the invention in well vascularized tumors. Compounds 5b, 6b and 6f were also tested in terms of feasibility of oral administration. Mice received a single dose of 40 mg/kg body weight of 5b, 6b or 6f, respectively. No signs of toxicity were observed. Then compounds 6b or 6f were given peroral as dual-dose application on two consecutive days in accordance to the i.p. treatment scheme used for the regression trials but with a 2-fold higher dose of 2x40mg/kg body weight. This was well tolerated and induced only a transient and marginal loss of body weight in case of 6b whereas no signs of toxicity were observed for 6f. In addition 6f was administered as single dose of 60 mg/kg body weight and again was tolerated symptom-free. This demonstrates the outstanding toxicity profile of the compounds of the present invention when given peroral even at higher doses.
To prove whether the tumor targeted cytotoxic activity is also achieved after an oral administration of compounds of the invention, the vascular disrupting effect was studied 24h after giving 60mg/kg body weight of compound 6b peroral. No signs of toxicity were observed. The vascular disrupting effect occurred to same extent as seen after i.p. application, as also shown in Figure 12.
Example 4: Cell growth inhibition assay in various human tumor cell lines
The effects of the compounds 5b, 6b, 8a, and 8e according to the present invention and of the reference compound 25f (Wang et aL, J. Med. Chem. 2002, 45, 1697-171 1 ) on cells of the following human tumor cell lines were evaluated: 518A2 melanoma, HL-60 leukemia, HT- 29 colon carcinoma, KB-V1/Vbl cervical carcinoma, and MCF-7/Topo breast carcinoma.
Figure imgf000056_0001
The HL-60 cells were obtained from the German Collection of Biological Material (DS Z), Braunschweig, Germany; the human 518A2 melanoma cells as well as the testicular germ cell tumor eel! lines H12.1 and 1411 HP were cultured in the department of onco!ogy and hematology, Medical Faculty of the Martin-Luther University, Halie, Germany; the KBV1/Vbl and the MCF-7/Topo cells were obtained from the Institute of Pharmacy of the University Regensburg, Germany; and the colon HT-29 cells from the University Hospital Erlangen, Germany. The HL-60 and the HT-29 cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 lU/mL penicillin G, 100 pg/mL streptomycin sulfate, 0.25 pg/mL amphotericin B and 250 pg/mL geniamycine (all from Gibco, Egenstein, Germany). The 518A2 and the KB-V1/Vbl cells were cultured in Dulbecco's Modified Eagle Medium (D-MEM, Gibco) containing 10% FCS, 100 lU/mL penicillin G, 100 pg/mL streptomycin sulfate, 0,25 pg/mL amphotericin B and 250 pg/mL gentamycine. The MCF-7/Topo cells were grown in E-MEM medium (Sigma) supplemented with 2.2 g/L NaHC03, 1 0 mg/L sodium pyruvate and 5% FCS. The cells were maintained in a moisture- saturated atmosphere (5% C02) at 37 °C in 75-mL culture flasks (Nunc, Wiesbaden, Germany). They were serially passaged following trypsinisation by 0.05% trypsin / 0.02% EDTA (PAA laboratories, Colbe, Germany). Mycoplasma contamination was routinely monitored, and only mycoplasma-free cultures were used.
MTT [3-(4,5-dimethy!thiazol-2-yl)-2,5-dipheny!tetrazolium bromide] (ABCR) was used to identify viable ceils which reduce it to a violet formazan (Mosmann, J. Immunol, Methods 1983, 65, 55-63). HL-60 leukemia cells (5x 05/mL), and cells (5x104/mL) of 518A2 melanoma, HT-29 colon, KB-VWbl cervix and MCF-7/Topo breast carcinoma were seeded out in 96-well tissue culture plates and cultured for 24 h. Incubation (5% CC½, 95% humidity, 37°C) of the cells following treatment with the test compounds was continued for 24, 48 or 72 h. Blank and solvent controls were treated identically. MTT in phosphate buffered saline (5 mg/mL) was added to a final concentration of 0.05% (HL-60, 518A2) or 0.1 % (HT-29, KB- V1 Vbl, MCF-7/Topo). After 2 h the formazan precipitate was dissolved in 10% sodium dodecylsulfate in DMSO containing 0.6% acetic acid in the case of the HL-60 cel!s. For the adherent 518A2, KB-V1/Vbi, CF-7/Topo and HT-29 ce!ls the micropiates were swiftiy turned to discard the medium before adding the solvent mixture. The micropiates were gently shaken in the dark for 30 min and absorbance at 570 nm and 630 nm (background) was measured with an ELISA plate reader. AN experiments were carried out in quadruplicate; the percentage of viable cells was calculated as the mean ± SD with controls set to 100%.
As shown in Figure 6A, the halo-amino substituted imidazoles 5b and 6b were distinctly more cytotoxic than the known reference compound 25f at iC5o concentrations in the single- digit nanomolar range, even in the combretastatin A-4 resistant HT-29 cells and the Pgp- overexpressing KB-V1 cells. Only in the MCF-7/Topo cells they were noticeably less active but still superior to the known imidazole 25f.
In contrast, the diamino substituted oxazole 8a and its imidazole counterpart 8e were more cell line specific with greater efficacy than 25f and 5b/6b against both multidrug-resistant cells, i.e., KB-V1/Vbl (Pgp+) and MCF-7/Topo (BCRP÷). This is a hint at potentially different or additional modes of action of these compounds.
Moreover, as shown in Figure 7, combretastatin A-4 shows lower cytotoxicities than compounds 5b and 6b in the multi-drug resistant HT-29 colon cancer cells. Hence, compounds 5b and 6b are able to overcome the drug resistance in these cancer cells in vitro.
The compounds 5b, 6b, 5f and 6f as well as the reference compound 25f (Wang et al.) were furthermore subjected to an SRB -cytotoxicity assay in a panel of tumor cell lines (H12.1 germ cell tumor, 141 1 HP germ ceil tumor, A2780 ovarian carcinoma, HT29 colon carcinoma, DLD1 colon carcinoma, HCT8 colon carcinoma). As shown in Figure 3, compounds 5b and 6b showed superior activity as compared to compound 25f, and a further improvement of cytotoxicity was observed for the compounds 5f and 6f.
The compounds 9a, 9b and 9c, which are exemplary compounds of formula (ill) according to the present invention, as weil as the reference compound 25f were tested in a similar manner. As indicated in Table 1 , compounds 9a, 9b and 9c showed a more pronounced and selective cytotoxic effect on tumor cells (L929 fibroblasts, KB-3-1 cervix carcinoma, and PC-3 prostata cancer) than on non-malign cells (PtK-2 opossum kidney cells and NHDF fibroblasts) as well as an improved effect (MIC) on HUVEC cells as compared to the reference compound 25f.
Figure imgf000058_0001
Table 1 : IC50 (nM) in L929 fibroblasts, KB-3-1 cervix carcinoma, PC-3 prostata cancer,
PtK-2 opossum kidney ce!ls and NHDF fibroblasts, and minimal inhibitory concentration (MIC) (nM) in HUVEC cells.
Example 5: Reactive oxygen species (ROS) generation assay
The generation of reactive oxygen species (ROS) in tumor cells is a sign of cellular stress resulting in severe damage of cellular components such as DNA, lipids or proteins. The amount of ROS produced in HL-60 and in 518A2 cells treated with the reference compound 25f or a compound according to the invention was assessed using the colorimetric nitroblue- tetrazolium (NBT) assay (Rook et al. J. Immunol. Methods 1985, 82, 161-167). The ROS generation (% NBT reduction) was determined from percent absorbance of formazan relative to untreated controls (1 %) after 24 h exposure of 518A2 and HL-60 cells to 50 μΜ of the test compounds. For the NBT assay, HL-60 cells (0.5'106/mL) were plated in 96-weli tissue culture plates, and test compounds were added after 24 h incubation at 37 °C to achieve a finai concentration of 50 μΜ. Incubation (5% C02, 95% humidity, 37°C) of cells following treatment with the test compounds was continued for 24 h. After removal of the cell medium by centrifugation, the cells in each well were resuspended in 100 pL 0.1 % NBT, and the plates were placed in the incubator for 1 h. The reduced NBT was solubilized with 100 μΙ 2 M KOH and 130 pi DMSO for 30 min. The absorbance was measured for each well at 630 and 405 nm (background) using an ELISA plate reader. The adherent 518A2 cells (0.5- 04/mL) were seeded out in 96- weil tissue culture plates after trypsinization and incubation for 24 h at 37 °C to allow attachment, then treated similarly, only that the medium was removed prior to incubation with NBT for 4 h. All experiments were carried out in quadruplicate. The following results were obtained, the indicated values representing means of four independent experiments ± standard deviation;
NBT Reduction [%] NBT Reduction [%]
in 518A2 cells after 24 h in HL-60 cells after 24 h
25f 2.2 ± 0.4 1.5 ± 0.1
5b 1.8 ± 0.2 4.4 ± 0.6
5c 1.6 ± 0.1 4.8 ± 0.5
5d 2.4 ± 0.4 6.0 ± 0.9
6b 1.5 ± 0.2 3.8 ± 0.3
6c 1.4 ± 0.3 3.9 ± 0.1
6d 1.6 ± 0.3 6.7 ± 0.8
8a 1.5 ± 0.1 3.5 + 0.4
8b 1.4 ± 0.3 3.3 ± 0.2
8c 1.1 ± 0.0 1.8 ± 0.3
8d 1.2 ± 0.1 1.4 ± 0.2
8e 1.6 + 0.3 6.4 ± 0.3 The weakly cytotoxic compound 25f led to a low ROS level, while, e.g., the highly cytotoxic compound 5b according to the invention afforded an ROS ievei of 4.4 %.
Example 6: Mitochondrial membrane assay
The extent of apoptosis-reiated mitochondrial damage in 518A2 and HL-60 cells was ascertained by means of the fluorescence dye JC-1 that detects changes in the mitochondrial membrane potential (Desager et aS. J. Cell. Biol. 1999, 144, 891-901 ). The ratio of red to green fluorescence relative to untreated controls (100%) after 72 h exposure of 518A2 and HL-60 ceils to 5 μΜ of the test compounds (i.e., compounds according to the invention or reference compound 25f) was determined with the Mitochondrial Membrane Detection Kit (Stratagene, La Jotla, CA, USA). Values represent means of four independent experiments ± standard deviation.
Changes in mitochondrial membrane potential were determined by the Mitochondrial Membrane Detection Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's procedure. 72 h following treatment with 5 μΜ of the test compounds, cell samples were centrifuged at 400 g for 5 min. The pellets were resuspended in 500 μ!_ diluted JC-1 solution, incubated at 37 °C for 15 min (HL-60) and then centrifuged again for 5 min at 400 g. After washing, the peilets were resuspended in 100 pL PBS and transferred into the wells of a black 96-well plate. The red (Aex = 585 nm, Kem = 590 nm) and green (Aex = 510 nm, Aem = 527 nm) fluorescence intensities were measured and their ratio was calculated.
Again, the impact of the test compounds was more diverse and specific in the HL-60 leukemia cells: intact mitochondria [%] Intact mitochondria [%]
in 518A2 cells after 72 h in HL-60 ceils after 72 h
25f 87 ± 7 74 ± 13
5b 80 ± 9 65 ± 9
5c 84 ± 6 55 ± 7
5d 80 ± 7 61 ± 13
6b 80 ± 9 66 ± 10
6c 89 ± 10 60 ± 15
6d 82 ± 7 63 ± 13
8a 85 ± 7 62 ± 9
8b 82 ± 8 62 ± 12
8c 94 ± 7 76 ± 19
8d 98 ± 15 90 ± 22
8e 77 ± 8 60 ± 15
Here, only about 65% of the mitochondria were intact after incubation with the more cytotoxic compound 5b, while treatment with 25f left more mitochondria unaffected (74%).
Example 7: TdT-mediated dUTP Nick-End Labelling (TUNEL) assay
!n TUNEL assays, which allow the detection of apoptosis by iabel!ing the 3'-OH ends of DNA fragments with fiuorescein-tagged nucleotides, the compounds 5b, 6b and 8a were found to induce death in HL-60 cells predominantly in an apoptotic way (about 60% after 16 h incubation with 10 μΜ drug). The extent of apoptosis-related mitochondrial damage in HL-60 cells was ascertained by means of the fluorescence dye JC-1 that detects changes in the mitochondrial membrane potential (Desager et a!., J. Cell. Biol. 1999, 144, 891-901 ). Only about 60% of the mitochondria were intact after incubation with the more cytotoxic compounds 5b-d, 6b-d and 8a/e, while treatment with reference compound 25f left 74% of the mitochondria unaffected. Microscopic images of HL-60 cells tested in TUN EL assays after incubation with the compound 5b, 6b or 8a are shown in Figure 8. A modification of the TdT-mediated dUTP Nick-End Labelling (TUNEL; Roche) assay described by Jobmann was used (Jobmann, M. Apoptose bei struktureiien Herzmuske!erkrankungen. Ph.D. Thesis, University Marburg, Germany 2002, 31 ). HL-60 ceils were incubated with the test compounds for 16 h, aliquots of 3 x 106 cells were withdrawn and washed/centrifuged 3 times in 200 pL PBS. The cells were fixed for 10 min at room temperature by suspending in 200 pL of a freshly prepared solution of 2% formalin in PBS, After washing with 2 x 200 pL PBS, 10 pL of the cell suspension was applied onto a microscope slide and air-dried at room temperature. The ceils were washed by covering with PBS for 5 min and treated for 2 min with a solution of 0.1 % Triton X-100 in 0.1% sodium citrate on ice. After washing two times with PBS, 10 pL of the freshly prepared TUNEL reaction mixture, consisting of 1 pL TUNEL-Enzyme solution and 9 pL TUNEL-Label solution, was dropped on the cells which were then covered and incubated (5% C02, 95% humidity) in the dark at 37 °C for 45 min. The cells were washed three times with PBS and then analysed by fluorescence microscopy at an excitation wavelength of 450-500 nm. The percentage of apoptotic, TUNEL-posttive, green-stained cells was counted and calculated for 300 cells and expressed as mean ± S.D. of three independent experiments.
Example 8: CAM assay Compounds of the present invention were tested for anti-angiogenic and vasculature disrupting properties using the CAM assay. In this test the vascular system of a fertilized chicken embryo is used as a model (Wilting et a!., Anat. Embryol. 1991 , 183, 259-271 ).
Fertilized chicken eggs received from a nearby farm directly after laying have been incubated at a temperature of 36-38 °C and a relative humidity of 60%. During the growth the eggs have been held in an inclined position and turned from time to time in order to avoid an adherence to the shell. After four days each embryo was transferred into a cavity created by fixing a thin plastic foil on top of a cup and covered. This was done by opening the shell at the flat end where the air sac resides and letting the content slip out. There the growth continued for another 2-4 days until the first blood vessels became visible. Then 10 nmol of the substance to be tested (in 10 pL PBS with 1 % DMF) have been applied directly on the embryonal vessels. As a reference PBS was used. Finally incubation continued for up to another three days (Dugan et al., Anat Rec. 1991 , 229, 125-128; Fisher, Tested studies for laboratory teaching 1993, 5, 105-1 15).
In Figure 9 the effects of the compounds CA-4 (reference) and 5b on the development of embryonal blood vessels compared to a negative control (PBS) are shown. Like CA-4, compound 5b led to a dramatic vessel shrinkage within 24 h after treatment and to a complete degradation of the vascular system within three days.
Example 9: Tube formation assay
The effect of the compounds 5b, 6b and 8a according to the invention as well as reference compound 25f (Wang et al., J. Med. Chem. 2002, 45, 1697-1711 ) on capillary tube formation in HUVEC cells on matrigel has been determined in order to further evaluate the anti- angiogenic properties of these compounds (the structures of the compounds employed are shown in Example 4).
35 μ!_ of media : matrigel (1 :1 ) were added into wells of a 96-well plate and incubated at 37 °C for 30 minutes. A trypsinized HUVEC cell suspension was set to 800.000 ceils/mL. In another 96-well plate 25 μΙ_ of the compound stock were serially diluted with 25 μΙ_ of media. 25 μΙ_ of compound dilutions and 25 μ!_ of cell suspension were added to the matrigel coated 96-well plate and the cells were incubated over night. As a negative control, methanol was used as in place of the compound dilutions. Compounds 6b and 8a showed pronounced inhibition of tube formation by HUVEC cells at very low concentrations (7.72 ng/mL), while compound 5b did not yield a correspondingly strong inhibition at this concentration, as also shown in Figure 14. Compounds 6b and 8a were distinctly more active than the known compound 25f, which inhibits tube formation at a much higher concentration (130 ng/mL).
Example 10: High-content analyses
Automatic microscopy based cluster analyses (High-Content Analyses, HCA) of the compounds 6b and 8a in PtK-2 cells revealed a close relationship to the known tubulin binder vinblastine for compound 6b and to the known PI3- inase inhibitor LY294002 for compound 8a. Results are shown in Figure 15. Instrumentation: ImageXpress Micro (IXM) High-Speed Laser Autofocus Digital CCD-camera
300 Watt Xenon arc lamp
Filter Sets: - DAPI
- FSTC
. TRITC
- Texas Red
Nikon Objectives: - 4X Plan Apo, NA 0,20
- 10X S Fluor, NA 0,50
- 20X S Fluor, NA 0,75
- 40X Plan Apo, NA 0,95
- 60X Plan Fluor, NA 0,85
Software: - MetaXpress
- AcuityXpress
Parameters (dye-/antibody assays): > 50, organised in modules, e.g., MWCS-moduie using DAP! (W1 ), FITC (W2) and TRSTC (W3) staining and filters. Descriptors of MWCS-module:
Image-based analysis: total celis, (%) positive W2 W3, scoring profile 1-/12-/1-3/123. absolute number of celis appearing stained only at wavelengths 1 and 2 but not 3 (12-) Cell-based analysis: total area (area of nuclei), stained area W1/W2/W3 (stained area for individual dyes), positive W2/W3 (absolute numbers of stained celis at each wavelength); average/integrated intensity W1/W2 W3
Reference compounds: 62
Cell lines: PtK2 (non-malignant), KB-3-1 cervix, A-498 kidney
carcinoma

Claims

Claims
1. A compound of formula (!)
Figure imgf000064_0001
(I) wherein:
X is selected from O, S, N(H), or N(C -4 a R1 is selected from halogen, -CN, -CF3
alkyl);
R2 is selected from hydrogen, halogen, -CN, -CF3i -OH, -0(Ci_4 alkyl), -NH2, -NH(C^ alkyl), or -N(d_4 alky!XC^ alkyl);
R3 is selected from -OH, -0(CM alkyi), -SH, -S(d^ alkyl), - H2, -NH(C^ aikyl), or -N C^ alkyl)(C1_4 alkyl); or, alternativefy, R2 and R3 jointly form a group -C(haiogen)=CH-N(CH3)-; or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
2. The compound of claim 1 , wherein R1 is selected from -CI, -Br, or -NH2.
3. The compound of daim 1 or 2, wherein Rz is selected from hydrogen, halogen, ~OH, or -NH2.
4. The compound of any of ciaims 1 to 3, wherein R3 is selected from -0-CH3, -0-CH2- CH3, or -N(CH3)2.
5. The compound of any of claims 1 to 4, wherein R3 is selected from -0-CH3 or -N(CH3)2.
6. The compound of claim 1 or 2, wherein R2 and R3 jointly form a group -C(CI)=CH- N(CH3)-.
7. The compound of any of ciaims 1 to 6, wherein X is selected from O and N(CH3).
8. The compound of claim 1 , wherein the compound is selected from 1-methyl-5-(3- amino-4-methoxyphenyl)-4-(3-chloro-4,5-dimethoxyphenyl)-imidazole, 1-methyl-5-(3- amino-4-methoxyphenyl)-4-(3-bromo-4,5-dimethoxyphenyl)-!midazole, 1-methyl-5-(3- amino-4-ethoxyphenyl)-4~(3-chloro-4,5-dimethoxyphenyl)-imidazole or 1 -methyl-5-(3- amino-4-ethoxyphenyl)-4-(3-bromo-4,5-dimethoxyphenyl)-imsdazole, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
9. The compound of claim 1 , wherein the compound is selected from 1-methyl-4-{3- chloro-4,5-dimethoxyphenyl)-5-(3-fluoro-4-ethoxyphenyl)-imidazole or 1 -methyl-4-(3- bromo»4,5-dimethoxypheny!)-5-(3-fluoro-4-ethoxyphenyl)-imidazoie, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
10. The compound of claim 1 , wherein the compound is selected from 1 -methyl-4-(3- amino-4,5-dimethoxyphenyl)-5-(N-methyl-3-chloroindol-5-yi)-imidazole, 1-methyi-4-{3- chloro-4,5-dimethoxyphenyI)-5-(N-methyl~3-chloroindol-5-y[)-imidazole or 1 -methyl-4- (3-bromo-4,5-dimethoxyphenyl)-5-(N-methyl-3-chloroindol-5-yl)-imidazole, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
1 1. A pharmaceutica! composition comprising the compound of any of claims 1 to 10 and a pharmaceutically acceptable excipient.
12. The compound of any of claims 1 to 10 or the pharmaceutical composition of claim 11 for use in treating or preventing cancer.
13. A method of treating or preventing cancer comprising administering a compound of any of claims 1 to 10 or a pharmaceutically acceptable salt, solvate, or prodrug thereof to a subject in need thereof.
14. The compound of claim 12, the pharmaceutical composition of claim 12 or the method of claim 13, wherein the cancer is selected from breast cancer, genitourinary cancer, lung cancer, gastrointestinal cancer, epidermoid cancer, melanoma, ovarian cancer, pancreas cancer, neuroblastoma, head and/or neck cancer, bladder cancer, renal cancer, brain cancer, leukemia, or lymphoma.
15. The compound of claim 12 or 14, the pharmaceutical composition of claim 12 or 14 or the method of claim 13 or 14, wherein the cancer is a multidrug-resistant cancer.
16. The compound of claim 12, 14 or 15, the pharmaceutical composition of claim 12, 14 or 15 or the method of claim 13, 14 or 15, wherein the cancer is resistant against combretastatin A-4 and/or cisp!atin.
17. The compound of any of daims 12, 14, 15 or 16, the pharmaceutical composition of any of claims 12, 14, 15 or 16 or the method of any of claims 13 to 16, whereby the compound or the pharmaceutical composition is to be administered in combination with an anti-proliferative drug, an anticancer drug, a cytostatic drug, a cytotoxic drug and/or radiotherapy.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2975029A1 (en) * 2014-07-18 2016-01-20 Charité - Universitätsmedizin Berlin 4,5-diarylimidazole derivatives as HDAC inhibitors
US9944597B2 (en) 2013-09-16 2018-04-17 The Board Of Regents Of The University Of Texas System Polysubstituted pyrroles having microtubule-disrupting, cytotoxic and antitumor activities and methods of use thereof
JP2018510857A (en) * 2015-03-02 2018-04-19 ライジェル ファーマシューティカルズ, インコーポレイテッド TGF-β inhibitor
EP3366303A1 (en) 2017-02-28 2018-08-29 Martin-Luther-Universität Halle-Wittenberg Enhancing the targeted in vivo delivery of cellular therapies

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102863388B (en) * 2011-07-05 2015-04-29 南京圣和药业股份有限公司 Tumor targeted drug Combretastatin A4 derivatives
SG11201608303QA (en) 2014-04-04 2016-11-29 Del Mar Pharmaceuticals Use of dianhydrogalactitol and analogs or derivatives thereof to treat non-small-cell carcinoma of the lung and ovarian cancer
CN104817519B (en) * 2015-05-11 2016-11-16 中国药科大学 The derivant of one class CA-4, its preparation method and medical usage thereof
CN108218855A (en) * 2018-03-12 2018-06-29 桑文军 A kind of novel tubulin inhibitor and its application in antitumor drug

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1071752A1 (en) 1998-04-21 2001-01-31 Micromet Gesellschaft für biomedizinische Forschung mbH CD19xCD3 SPECIFIC POLYPEPTIDES AND USES THEREOF
WO2001009103A2 (en) 1999-08-02 2001-02-08 Abbott Laboratories Imidazole antiproliferative agents

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1071752A1 (en) 1998-04-21 2001-01-31 Micromet Gesellschaft für biomedizinische Forschung mbH CD19xCD3 SPECIFIC POLYPEPTIDES AND USES THEREOF
WO2001009103A2 (en) 1999-08-02 2001-02-08 Abbott Laboratories Imidazole antiproliferative agents

Non-Patent Citations (33)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences"
BEDFORD ET AL., BIOORG. MED. CHEM. LETT., vol. 6, 1996, pages 157 - 160
BROWN, TOP. HETEROCYCL. CHEM., vol. 2, 2006, pages 1 - 51
BUNDGAARD, H.: "Design of Prodrugs", 1985, ELSEVIER, pages: 7 - 9,21-24
DESAGER ET AL., J. CELL. BIOL., vol. 144, 1999, pages 891 - 901
DESAGER, J. CELL. BIOI., vol. 144, 1999, pages 891 - 901
DUGAN ET AL., ANAT. REC., vol. 229, 1991, pages 125 - 128
FISHER, TESTED STUDIES FOR FABORATORY TEACHING, vol. 5, 1993, pages 105 - 115
FOLKES ET AL., CHEM. RES. TOXICOL., vol. 20, 2007, pages 1885 - 1894
HOLWELL ET AL., ANTICANCER RES., vol. 22, 2002, pages 3933 - 40
JOBMANN, M.: "Ph.D. Thesis", 2002, UNIVERSITY MARBURG, article "Apoptose bei strukturellen Herzmuskelerkrankungen", pages: 31
KISS ET AL.: "Discovery of a long-acting, peripherally selective inhibitor of catechol-O-methyltransferase", J. MED. CHEM., vol. 53, no. 8, 22 April 2010 (2010-04-22), pages 3396 - 3411, XP002594266 *
KISS LE ET AL., J MED CHEM, vol. 53, no. 8, 2010, pages 3396 - 411
LIPPERT, BIOORG. MED. CHEM., vol. 15, 2007, pages 605 - 615
MOSMANN, J. IMMUNOL. METHODS, vol. 65, 1983, pages 55 - 63
MUELLER ET AL., CANCER RES., 2003
MUELLER ET AL., TUMOR BIOL., 2006
MÜLLER ET AL.: "Failure of activation of caspase-9 induces a higher threshold for apoptosis and cisplatin resistance in testicular cancer", CANCER RES., vol. 63, 2003, pages 513 - 521
OHSUMI K ET AL., BIOORG MED CHEM LETT, vol. 8, no. 22, 1998, pages 3153 - 8
OHSUMI K ET AL: "Syntheses and antitumor activity of cis-restricted combretastatins: 5-membered heterocyclic analogues", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, PERGAMON, ELSEVIER SCIENCE, GB LNKD- DOI:10.1016/S0960-894X(98)00579-4, vol. 8, no. 22, 17 November 1998 (1998-11-17), pages 3153 - 3158, XP004143718, ISSN: 0960-894X *
ORSINI ET AL.: "Natural stilbenes and analoga as antineoplastic agents, in: Atta-ur-Rahman", STUDIES IN NATURAL PRODUCTS CHEMISTRY, BIOACTIVE NATURAL PRODUCTS, vol. 34, 2008
PAPAZISIS: "Optimization of the sulforhodamine B colorimetric assay", J. IMMUNOL. METHODS, vol. 208, 1997, pages 151 - 158
PETTIT ET AL., J. NAT. PROD., vol. 68, 2005, pages 1450 - 1458
ROOK ET AL., J. IMMUNOL. METHODS, vol. 82, 1985, pages 161 - 167
ROXANA ET AL., MAEDICA, vol. 1, no. 1, 2006, pages 63 - 65
TAYLOR ET AL.: "Antibody therapy for rheumatoid arthritis", CURR OPIN PHARMACAL, vol. 3, no. 3, 2003, pages 323 - 328
TAYLOR ET AL.: "Antibody therapy for rheumatoid arthritis", CURR OPIN PHARMACOL, vol. 3, no. 3, 2003, pages 323 - 328
TRON ET AL., J. MED. CHEM., vol. 49, 2006, pages 3033 - 3044
VASILEVSKY ET AL., CHEMISTRY OF HETEROCYCLIC COMPOUNDS, vol. 44, no. 10, 2008, pages 1257 - 1261
WANG ET AL., J. MED. CHEM., vol. 45, 2002, pages 1697 - 1711
WANG ET AL.: "Potent, orally active heterocycle-based combrestatin A-4 analogues: synthesis, structure-activity relationship, pharmacokinetics, and in vivo antitumor activity evaluation", J. MED. CHEM., vol. 45, 20 February 2001 (2001-02-20), pages 1697 - 1711, XP002304485 *
WEHNER, GEHRING: "Thieme Verlag", 1995, THIEME VERLAG
WILTING ET AL., ANAT. EMBRYOL., vol. 183, 1991, pages 259 - 271

Cited By (6)

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